Methods for treating inflammation

ABSTRACT

The present invention provides a method for treating inflammation in a subject which comprises administering to the subject soluble receptor for advanced glycation endproduct (sRAGE) in an amount effective to inhibit binding of advanced glycation endproducts (AGEs) to RAGE thereby treating inflammation in the subject. The present invention also provides for a method for treating inflammation in a subject which comprises administering to the subject an agent in an amount effective to inhibit the interaction between receptor for advanced glycation endproduct (RAGE) and its ligand thereby treating inflammation in the subject.

This application claims the priority of U.S. Ser. No. 08/755,235, filedNov. 22, 1996; and U.S. Ser. No. 08/948,131, filed Oct. 9, 1997; and PCTInternational Application No. PCT/US99/23303 which iscontinuation-in-part of U.S. Ser. No. 09/263,312, filed Mar. 5, 1999which is a continuation-in-part of U.S. Ser. No. 09/167,705, filed Oct.6, 1998, the contents of all of which are hereby incorporated byreference into this application.

The invention disclosed herein was made with Government support underGrant Nos. HL21006 and AG00603 from the National Institutes of Health,U.S. Department of Health and Human Services. Accordingly, the U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byauthor and date. Full citations for these publications may be foundlisted alphabetically at the end of each section of the ExperimentalDetails section of the application. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein.

SUMMARY OF THE INVENTION

The present invention provides a method for treating inflammation in asubject which comprises administering to the subject soluble receptorfor advanced glycation endproduct (sRAGE) in an amount effective toinhibit binding of advanced glycation endproducts (AGEs) to RAGE therebytreating inflammation in the subject.

BRIEF DESCRIPTION OF THE FIGURES Wound Healing

FIG. 1. Effect of sRAGE on wound healing in the genetically-diabeticdb+/db+ mouse. A full-thickness 1.5×1.5 cm wound was created on thebacks of db+/db+ mice or control, heterozygote db+/m+mice and coveredwith TEGADERM®. Diabetic wounds were treated with eitherphosphate-buffered saline (PBS) directly under the TEGADERM® daily for 7days commencing on day 3 following surgery or with sRAGE (200 ng). Woundarea was measured at baseline through day 21 by placing a glass slideover the wound area, tracing the wound area, and placing thisinformation into a computer in order to calculate the percentage ofwound closure as a function of time. Left axis represents percent woundclosure.

FIG. 2. Administration of sRAGE to the genetically-diabetic db+/db+mouseimproves wound healing: dose-response studies. Wounds were created asabove and treated from days 3 through 9 with sRAGE (either 2,000, 200,or 20 ng/day) or with phosphate-buffered saline. At day 10, wound areawas measured and compared with initial wound area as above. Results arepresented as fold increase in percent wound healing compared with micetreated with phosphate buffered saline (defined as one in figure). Allstatistical analyses are shown comparing wound healing in the presenceof different doses of sRAGE vs. treatment of diabetic wounds withphosphate-buffered saline.

FIGS. 3A and 3B. AGE-immunoreactive epitopes in the wounds of diabetic(db+/db+) mice. 1.5×1.5 cm full-thickness wounds created in the backs ofdiabetic mice (db+/db+ mice; FIG. 3A) and non-diabetic mice (db+/m+;FIG. 3B) were excised, fixed and sections stained with affinity-purifiedanti-AGE IgG. Magnification: 200×.

Periodontal Disease

FIG. 4. Measurements of alveolar bone loss in mice treated with sRAGE.Statistical analysis=Group I vs. Group II—p=0.002; Group I vs. GroupIII—p=0.005; Group III vs. Group IV: p=0.009.

Delayed Type Hypersensitivity

FIG. 5. Immunohistochemistry of human kidney (active lupus nephritis).Kidney tissue from a patient with active lupus nephritis was obtained,fixed in formalin and paraffin-embedded sections were prepared. Sectionswere stained with rabbit anti-RAGE IgG. Increased expression of RAGE wasnoted in the podocytes of the glomerulus.

FIG. 6. Incubation of HUVECs with EN-RAGE results in increased cellsurface VCAM-1. Human umbilical vein endothelial cells were cultured inserum-free RPMI 1640 without endothelial cell growth factor for 24 hrsand then stimulated with EN-RAGE or bovine serum albumin (BSA); both 10μg/ml. Where indicated, cells were pretreated with rabbit anti-humanRAGE IgG, nonimmune rabbit IgG; in certain cases, EN-RAGE was pretreatedwith the indicated concentration of soluble RAGE (sRAGE) for 2 hrs priorto stimulation with EN-RAGE. After eight hrs stimulation with EN-RAGE,cells were fixed as described above. Cell surface ELISA employinganti-VCAM-1 IgG was performed. Statistical considerations are shown inthe figure.

FIG. 7. Incubation of HUVECs with EN-RAGE increases VCAM-1 functionalactivity: increased binding of Molt-4 cells. Assessment of functionalVCAM-1 activity was determined using ⁵¹Cr-labelled Molt-4 cells (ATCC)as described above. HUVEC were treated with either BSA (10 μg/ml) orEN-RAGE (5 μg/ml) for eight hrs. Molt-4 cells (5×10⁷/ml) were incubatedfor 2 hrs in RPMI containing ⁵¹Cr (0.1 mCi). At the end of that time,cells were washed with PBS and then added to the monolayer of treatedHUVEC for one hour. Unbound Molt-4 cells were removed by washing threetimes with PBS. Cells were then lysed in buffer containing triton-X 100(2%) in order to release Molt-4 cell-bearing radioactivity. Statisticalconsiderations are shown in the figure.

FIG. 8. Delayed hypersensitivity model: suppression of inflammation inthe presence of soluble RAGE. CF-1 mice were sensitized with mBSA; afterthree weeks, mBSA was injected into the hind foot pad. Certain mice weretreated with the indicated concentrations of mouse serum albumin, sRAGEor the indicated F(ab′)₂ antibody fragments of RAGE or EN-RAGE.Inflammation score was defined as above (scale; 1-9).

FIG. 9. Nucleic Acid Sequence of bovine EN-RAGE. The cDNA for bovineEN-RAGE was cloned and deposited with Genbank at Accession No. AF011757. (SEQ ID NO:16).

Collagen-Induced Arthritis

FIG. 10. Identification of wild-type RAGE and (G82S) and (S82S)polymorphisms. Genomic DNA was prepared from whole blood of controls,and subjects with RA. Amplification of exon 3 of the RAGE gene wasperformed as described; the resulting PCR products were digested withAlu 1. Upon agarose gel electrophoresis, identification of wild-typeRAGE (G82G), and mutant RAGE (G82S) or (S82S) alleles was performed.

FIGS. 11A-11E. Transfection of CHO cells with mutant RAGE (82S) confersincreased affinity and cellular responsiveness to EN-RAGE. CHO cells,which endogenously do not express RAGE, were stably-transfected withpcDNA3.1 vector containing cDNA encoding wild-type human RAGE or mutant(82S) human RAGE. “Mock” controls indicated empty vector. FIG. 11A.Immunoblotting. Lysates of stably-transfected CHO cells were preparedand subjected to immunoblotting using anti-human RAGE IgG (2 μg/ml).FIGS. 11B-C. Radioligand binding assays. Purified EN-RAGE wasradiolabelled using ¹²⁵-I and radioligand binding assays were performedin 96-well tissue culture dishes containing the indicated transfectedCHO cells. Assays were performed in the presence of the indicatedconcentration of radiolabelled EN-RAGE±an 50-fold molar excess ofunlabeled EN-RAGE. Elution of bound material was performed in a solutioncontaining heparin. Equilibrium binding data were analyzed according tothe equation of Klotz and Hunston. Where indicated, pretreatment witheither antibodies (70 μg/ml), human soluble RAGE or bovine serum albumin(50-fold molar excess) was performed. The mean ±standard deviation (SD)is shown. In FIG. 1C, * indicates p<0.01 versus respective controls.FIG. 11D. Activation of p44/p42 MAP kinases. The indicatedstably-transfected CHO cells were incubated with EN-RAGE, 10 μg/ml, forone hr. Cell lysates were subjected to SDS-PAGE and transfer of thegels' contents to nitrocellulose. Immunblotting was performed usinganti-phosphorylated p44/p42 MAP kinase (1 μg/ml). Where indicated,pretreatment with either BSA or sRAGE (50-fold molar excess), or theindicated IgG (70 μg/ml), for 2 hrs was performed. Controlimmunoblotting using antibody to total p44/p42 MAP kinase revealed thatthere were no differences in levels of total p44/p42 in each group (notshown). FIG. 11E. Activation of NF-kB. Nuclear extracts were preparedfrom the indicated stably-transfected CHO cells incubated with EN-RAGE,10 μg/ml, for 6 hrs, and EMSA was performed. Where indicated, cells werepretreated with either nonimmune/anti-RAGE IgG (70 μg/ml), soluble RAGEor BSA (50-fold molar excess) for 2 hrs prior to incubation withEN-RAGE. In FIGS. 11D-11E, bands were scanned into a densitometer, andband density was quantified using ImageQuant. These experiments wereperformed at least three times, and representative experiments areshown.

FIGS. 12A-F. Human peripheral blood mononuclear phagocytes (Mps)expressing RAGE (G82S) or (S82S) display increased responsiveness toEN-RAGE. MPs were purified from the blood of human subjects expressingwild-type RAGE, (G82S), or (S82S). For these experiments, MPs from 8wild-type RAGE-bearing subjects, and 8 subjects bearing RAGE (G82S) or(S82S) were employed. FIGS. 12A-B. Activation of p44/p42 MAP kinases.The indicated MPs were incubated with EN-RAGE, 10 μg/ml, for one hr, orwith no mediator. Cell lysates were prepared and immunoblottingperformed using anti-phosphorylated p44/p42 MAP kinase. Densitometricanalysis was performed and is shown in FIG. 12B. Since multipleexperiments demonstrated that there were no differences in the extent ofcellular activation in MPs bearing (G82S) or (S82S), these two groupswere combined for analyses. Control immunoblotting using antibody tototal p44/p42 MAP kinase revealed that there were no differences inlevels of total p44/p42 in each group. The mean ±SD is shown. FIGS.12C-D. Generation of TNF-alpha (FIG. 12C) and IL-6 (FIG. 12D). Human MPsbearing the indicated RAGE alleles were cultured in the presence ofeither no mediator, or EN-RAGE, 10 μg/ml, for 14 hrs. Supernatants wereretrieved and levels of TNF-alpha and IL-6 determined by ELISA. The mean±SD is shown. FIGS. 12E-F. Activity of MMP-9. Human MPs bearing theindicated RAGE alleles were cultured in the presence of either nomediator, or EN-RAGE, 10 μg/ml, for 14 hrs. Supernatants were retrievedand subjected to zymography to assess levels of activated MMP-9. Bandswere scanned into a densitometer and band density was quantified. Themean ±SD is shown. In FIGS. 12B, C, D and F, * indicates p<0.01 versusbaseline. Other statistical comparisons are indicated.

FIGS. 13A-J. Induction of arthritis by bovine type II collagen in dba/1mice enhances expression of RAGE and EN-RAGEs. Dba/1 mice wereimmunized/challenged with bovine type II collagen. Control mice were nottreated. Six weeks after immunization, joint tissue from the hind feet(FIGS. 13A-H) or stifle joint (FIGS. 13I-J) was prepared for study.FIGS. 13A-H. Histology. In FIGS. 13A-B, tissue was subjected to H&Eanalysis. Immunohistochemistry using anti-RAGE IgG (30 μg/ml) (FIGS. 13C-D); anti-EN-RAGE IgG (3 μg/ml) (FIGS. 13E-F); or rabbit nonimmune IgG(30 μg/ml) (FIGS. 13G-H) was performed. Scale bar: 300 μm. FIGS. 13I-J.Immunoblotting. Lysates were prepared from stifle joints and subjectedto immunoblotting using anti-RAGE IgG (4.7 μg/ml) (FIG. 13I); oranti-EN-RAGE IgG (2 μg/ml) (FIG. 13J). For each group, n=3 mice percondition; representative bands are shown. Densitometric analysis ofband intensity was performed, and the mean ±SD is shown.

FIGS. 14A-F. Blockade of RAGE suppresses development of arthritis andmarkers of inflammation in dba/1 mice immunized/challenged with bovinetype II collagen. FIGS. 14A-B, Clinical scoring. In FIG. 14A, at theindicated time points after immunization/challenge with bovine type IIcollagen and treatment with vehicle, murine serum albumin (MSA) orsRAGE, hind foot pad thickness was measured with calipers. The mean±standard deviation (SD) is shown; n=10 mice per group. * indicatesp<0.001. In FIG. 14B, clinical scoring of wrist joint redness/swellingwas performed by a blinded observer 6 weeks after immunization withbovine type II collagen. The mean ±SD is shown; n=10 mice per group. *indicates p=0.0001. FIGS. 14C-F. Assessment of markers of inflammation.FIGS. 14C-D. TNF-alpha. In FIG. 14C, stifle joint tissue of mice withcollagen-induced arthritis was retrieved six weeks after immunizationwith bovine type II collagen. Lysates were subjected to immunoblottingusing anti-murine TNF-alpha IgG (1 μg/ml). In FIG. 14D, plasma from micewith collagen-induced arthritis was subjected to ELISA for levels ofTNF-alpha. In FIG. 14C, 3 mice per group; and in FIG. 14D, 10 mice pergroup, were employed. The mean ±SD is shown. In FIG. 14C, * indicatesp=0.001; and in FIG. 14D, * indicates p=0.03. FIGS. 14E-F. IL-6 andIL-2. Stifle joint tissue was retrieved from control mice (clear bars)and mice with collagen-induced arthritis (black bars). Lysates wereprepared and ELISA was performed for determination of levels of IL-6(FIG. 14E) and IL-2 (FIG. 14F). Results are reported as ng/μg tissue.The mean ±SD is shown; n=6 mice per group. In FIG. 14E, * indicatesp=0.04.

FIGS. 15A-D. Blockade of RAGE suppresses generation of MMPs in dba/1mice immunized/challenged with bovine type II collagen. FIGS. 15A-B.Immunoblotting. Stifle joint tissue was retrieved from control mice(clear bars) and mice with bovine type II collagen-induced arthritis(black bars) six weeks after initial immunization. Lysates were preparedand subjected to immunoblotting using either anti-MMP-2 IgG (FIG. 15A)or anti-MMP-9 IgG (FIG. 15B). The mean ±SD is shown; n=3 mice per group.FIGS. 15C-D. Zymography. Lysates were prepared from stifle joint tissueof control mice, or mice with collagen-induced arthritis. Zymography wasperformed and the results subjected to densitometric analysis. The mean±SD is shown; n=3 mice per group.

Statistical analyses: MMP-2: * indicates p=0.001 vs control; and p=0.004vs sRAGE. MMP-9: * indicates p=0.02 vs control; and p=0.005 vs sRAGE.

FIGS. 16A-B. Blockade of RAGE suppresses extra-articular inflammatoryresponses induced by bovine type II collagen.

FIG. 16A Ear swelling. Six weeks after immunization with bovine type IIcollagen, MSA- and sRAGE-treated mice were injected with bovine type IIcollagen (10 μg) into ear tissue. Ear thickness was measured withcalipers by a blinded observer immediately prior to local injection, and18 hrs later. The mean ±SD is shown; n=5 mice per group. FIG. 16B.Splenocyte proliferation. Splenocytes were prepared from the indicatedmice at sacrifice, 6 weeks after immunization. Baseline levels ofsplenocyte proliferation, and proliferation in the presence of bovinetype II collagen or PMA (0.1 μg/ml in each case) was determined. Notethat no additional MSA or sRAGE was added to the culture system. Themean ±SD is shown; n=5 mice per group.

Autoimmune Diseases, EAE

FIG. 17. Life Table analysis of development of diabetes in NOD.SCIDrecipients with and without treatment with sRAGE. A single experimentrepresentative of 3 is presented. Soluble RAGE treatment reduced thedevelopment of diabetes in the NOD.SCID recipients. In pooledexperiments (n=12 in sRAGE treated and n=13 controls, the incidence ofdiabetes at 35 days was 17% and 92%, respectively; p<0.001).

FIG. 18. Histology of islets in control and sRAGE treated recipients ofsplenocytes (H&E staining).

FIG. 19. Immunostaining for TNF-a and IL-1β in the islets ofsRAGE-treated and control mice. Pancreases from untreated (control) orsRAGE-treated recipient mice were stained with antibodies to TNF (a) orIL-1β (b). Expression of these inflammatory cytokines (dark brown) wasreduced in sRAGE-treated mice and, in the latter, was predominantly inthe area of peri-insulitis.

FIG. 20. Symptomatic scoring of EAE in B10.PL mice immunized withMBP-derived peptide and treated with vehicle (mouse serum albumin; 50μg/day; fatty acid-free, Sigma) or sRAGE (50 μg/day).

FIGS. 21A-21D. Histologic analysis (H&E) of spinal cord from miceimmunized with MBP and treated with vehicle (FIG. 21B; the animal wassacrificed on day 21 with full-blown symptomatic EAE) or sRAGE (FIG.21C; the animal was sacrificed on day 35 and was asymptomatic) as inFIG. 20. FIG. 21A shows normal mouse spinal cord. FIG. 21D showsquantitation of nuclei in affected areas (this reflects principallycells in the inflammatory infiltrate).

FIG. 22. Symptomatic analysis of B10.PL mice infused with activated1AE10 cells as described in the text. As indicated, mice received eitheranti-RAGE IgG or nonimmune IgG for 15 days (see text for detailsexperimental protocol).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating inflammation in asubject which comprises administering to the subject an amount ofsoluble receptor for advanced glycation endproducts (sRAGE) effective totreat inflammation in the subject.

The present invention also provides a method for treating inflammationin a subject which comprises administering to the subject atherapeutically effective amount of an agent which inhibits binding ofadvanced glycation endproducts (AGEs) to any receptor for advancedglycation endproducts so (RAGE) as to treat inflammation in the subject.The advanced glycation endproduct (AGE) may be a pentosidine, acarboxymethyllysine, a carboxyethyllysine, a pyrallines, an imidizalone,a methylglyoxal, an ethylglyoxal.

The present invention also provides a method for treating arthritis in ahuman subject which comprises administering to the subject atherapeutically effective amount of an agent which inhibits binding ofadvanced glycation endproducts (AGEs) to any receptor for advancedglycation endproducts so (RAGE) as to treat arthritis in the subject.

Inflammation in the subject may be associated with any one or more ofvarious conditions or diseases. For example, the inflammation in thesubject may be due to a wound, to periodontal disease, to delayed-typehypersensitivity, to autoimmune disease, or to arthritis. The subjectmay be suffering from an autoimmune disease. In one embodiment, thesubject is suffering from multiple sclerosis, autoimmune encephalitis,lupus nephritis, or autoimmune complications from diabetes. The subjectmay be suffering from diabetes, for example, type I diabetes. Thesubject may be suffering from Behchet's syndrome. The subject may besuffering from Sjogren's syndrome. The subject may be suffering fromcolitis, ulcerative colitis, inflammatory colitis, Crohn's disease orthe like. The subject may be suffering from arthritis which isosteoarthritis, rheumatoid arthritis, collagen-induced arthritis,psoriatic arthritis, lupus-induced arthritis, or trauma-inducedarthritis. The subject may be suffering from another overall conditionor disease which includes a manifestation of inflammation, such asarthritis. In another embodiment, the subject is suffering from anallergy or is experiencing an allergic response. In one embodiment, thesubject is suffering from asthma. The subject may be suffering fromallergic asthma. In another embodiment, the subject is suffering fromsystemic lupus erythematosus, inflammatory lupus nephritis, septic shockor endotoxemia.

In a further embodiment, the subject is suffering from an autoimmune orinflammatory disorder in which recruitment of EN-RAGE-containinginflammatory cells occurs. In another embodiment, the subject issuffering from a bacterial-associated or other pathogen-associatedinfection.

The inflammation to be treated, in one embodiment of the invention, iscaused by the accumulation of the AGEs in certain tissues dependent uponthe ongoing biology in a subject. For example, the lesions in the bloodvessels which can occur in a subject suffering from diabetes are due tothe increased accumulation of AGEs in the presence of higher sugar inthe blood. Therefore, the administration of the agent as describedherein would reduce the interaction between the AGEs in the blood andthe receptor for AGE, thereby reducing inflammation at that site.Therefore, the present invention encompasses inflammation which wouldoccur in a subject at locations where the AGE products accumulate due tothe overriding disease or condition.

The present invention also provides for a method for inhibitingperiodontal disease in a subject which comprises administering topicallyto the subject a pharmaceutical composition which comprises sRAGE in anamount effective to accelerate wound healing and thereby inhibitperiodontal disease. The pharmaceutical composition may comprise sRAGEin a toothpaste.

The present invention provides for a new proinflammatory cytokine-likemolecule (EN-RAGE) (which has some sequence similarity to the family ofcalgranulin molecules). EN-RAGE is a protein located inside ofinflammatory cells (such as neutrophils) and which may be released bysuch inflammatory cells. EN-RAGE has biological activity that may beresponsible for the propagation and sustainment of an inflammatoryresponse by interacting with cellular receptor RAGE.

The subject on which any of the methods of the invention is employed maybe any mammal, e.g. a human subject, a murine subject, a bovine subject,a porcine subject, a canine subject, a primate subject, a felinesubject, etc. Preferably, the subject is a human subject. However, formethods of identifying a compound or agent which is useful in treatinginflammation, preferably, the subject is a primate, or murine subject.

The cell may be a eukaryotic cell. The cell may be a cell of a subject.The subject may be a human. The cell may be a neuronal cell, anendothelial cell, a glial cell, a microglial cell, a smooth muscle cell,a somatic cell, a bone marrow cell, a liver cell, an intestinal cell, agerm cell, a myocyte, a mononuclear phagocyte, an endothelial cell, atumor cell, a lymphocyte cell, a mesangial cell, a retinal epithelialcell, a retinal vascular cell a ganglion cell or a stem cell. The cellmay also be other kinds of cells not explicitly listed herein. The cellmay be any human cell. The cell may be a normal cell, an activated cell,a neoplastic cell, a diseased cell or an infected cell.

The Agent

In accordance with the method of this invention, the agent may comprisea polypeptide, a peptidomimetic, an organic molecule, a carbohydrate, alipid, an antibody or a nucleic acid. The polypeptide may be synthesizedchemically or produced by standard recombinant DNA methods. Inaccordance with the method of this invention, the polypeptide maycomprise an advanced glycation endproduct polypeptide or a portionthereof, a receptor for an advanced glycation endproduct polypeptide ora portion thereof, a soluble receptor for advanced glycation endproductpolypeptide (sRAGE) or a portion thereof. In one embodiment of theinvention, the portion of sRAGE is the V-domain of RAGE, which is theamino terminal 112 amino acids (not including the leader peptide).

The sequence of the V-domain of mature human RAGE is the following:

(SEQ ID NO:1) Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu Val LeuLys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu Trp Lys Leu AsnThr Gly Arg Thr Glu Ala Trp Lys Val Leu Ser Pro Gln Gly Gly Gly Pro TrpAsp Ser Val Ala Arg Val Leu Pro Asn Gly Ser Leu Phe Leu Pro Ala Val GlyIle Gln Asp Glu Gly Ile Phe Arg Cys Gln Ala Met Asn Arg Asn Gly Lys GluThr Lys Ser Asn Tyr Arg Val Arg Val Tyr Gln Ile Pro Gly Lys Pro Glu IleVal Asp Ser Ala Ser Glu Leu Thr.

The sequence of mature human RAGE not including the 22 amino acid leadersequence is:

(SEQ ID NO:2) Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu Pro Leu Val LeuLys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu Trp Lys Leu AsnThr Gly Arg Thr Glu Ala Trp Lys Val Leu Ser Pro Gln Gly Gly Gly Pro TrpAsp Ser Val Ala Arg Val Leu Pro Asn Gly Ser Leu Phe Leu Pro Ala Val GlyIle Gln Asp Glu Gly Ile Phe Arg Cys Gln Ala Met Asn Arg Asn Gly Lys GluThr Lys Ser Asn Tyr Arg Val Arg Val Tyr Gln Ile Pro Gly Lys Pro Glu IleVal Asp Ser Ala Ser Glu Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr CysVal Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser Trp His Leu Asp Gly LysPro Leu Val Pro Asn Glu Lys Gly Val Ser Val Lys Glu Gln Thr Arg Arg HisPro Glu Thr Gly Leu Phe Thr Leu Gln Ser Glu Leu Met Val Thr Pro Ala ArgGly Gly Asp Pro Arg Pro Thr Phe Ser Cys Ser Phe Ser Pro Gly Leu Pro ArgHis Arg Ala Leu Arg Thr Ala Pro Ile Gln Pro Arg Val Trp Glu Pro Val ProLeu Glu Glu Val Gln Leu Val Val Glu Pro Glu Gly Gly Ala Val Ala Pro GlyGly Thr Val Thr Leu Thr Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile HisTrp Met Lys Asp Gly Val Pro Leu Pro Leu Pro Pro Ser Pro Val Leu Ile LeuPro Glu Ile Gly Pro Gln Asp Gln Gly Thr Tyr Ser Cys Val Ala Thr His SerSer His Gly Pro Gln Glu Ser Arg Ala Val Ser Ile Ser Ile Ile Glu Pro GlyGlu Glu Gly Pro Thr Ala Gly Ser Val Gly Gly Ser Gly Leu Gly Thr Leu AlaLeu Ala Leu Gly Ile Leu Gly Gly Leu Gly Thr Ala.

The agent may be a composition which consists essentially of sRAGE. Theagent may be a polypeptide which is fragment of sRAGE, for example, afragment which is the V-domain of sRAGE.

In several embodiments of the present invention, the agent is a peptidehaving an amino acid sequence corresponding to the amino acid sequenceof a V-domain of a RAGE or soluble RAGE is exemplified by the followingamino acid sequences:

(SEQ ID No:3) A-Q-N-I-T-A-R-I-G-E-P-L-V-L-K-C-K-G-A-P-K-K-P-P-Q-R-L-E-W-K; (SEQ ID NO:4) G-Q-N-I-T-A-R-I-G-E-P-L-V-L-S-C-K-G-A-P-K-KP-P-Q- Q-L-E-W-K; (SEQ ID NO:5)G-Q-N-I-T-A-R-I-G-E-P-L-M-L-S-C-K-A-A-P-K-K-P-T-Q- K-L-E-W-K; (SEQ IDNO:6) D-Q-N-I-T-A-R-I-G-K-P-L-V-L-N-C-K-G-A-P-K-K-P-P-Q- Q-L-E-W-K.

The present invention provides for an isolated peptide having an aminoacid sequence which corresponds to the amino acid sequence of the first1-112 amino acids of human RAGE (which is the V-domain of human RAGE),or which corresponds to amino acids 5-35 of the V-domain of human RAGE,or any other smaller portion of the V-domain of human RAGE.Representative peptides of the present invention include but are notlimited to peptides having an amino acid sequence which corresponds toamino acid numbers (2-30), (5-35), (10-40), (15-45), (20-50), (25-55),(30-60), (30-65), (10-60), (8-100), 14-75), (24-80), (33-75), (45-110)of human sRAGE protein.

The agent or inhibitor of the present invention may comprise a peptidehaving an amino acid sequence corresponding to amino acid numbers 1-30of the V-domain of sRAGE (soluble receptor for advanced glycationendproducts). The sRAGE may be human, mouse, rat or bovine sRAGE.

The agent may be a peptide, a peptidomimetic, a nucleic acid or a smallmolecule. The terms “peptide” and “polypeptide” are used interchangablythroughout. The peptide may be at least a portion of the sequence fromamino acid 1 to amino acid 30 of sRAGE. The peptide may be a peptideconsisting essentially of the amino acid sequence of SEQ ID NOS: 1, 2,3, 4, 5, or 6. The peptide may be smaller than SEQ ID NO:1, retainingamino acid regions necessary to mimic the binding site of sRAGE. Thepeptide may comprise amino acids 1-112 of a RAGE protein (not includingthe leader sequence), i.e. the V-domain. The peptide may consistessentially of the V-domain of a RAGE protein (SEQ ID NO:1).

The polypeptide may be a peptidomimetic, a synthetic polypeptide or apolypeptide analog. The polypeptide may be a non-natural polypeptidewhich has chirality not found in nature, i.e. D-amino acids or L-aminoacids.

The polypeptide may be a derivative of a natural polypeptide, a modifiedpolypeptide, a labelled polypeptide, or a polypeptide which includesnon-natural peptides. The peptidomimetic may be identified fromscreening large libraries of different compounds which arepeptidomimetics to determine a compound which is capable of inhibitinginteraction of an amyloid β peptide with a receptor for advancedglycation endproduct. In another embodiment, the polypeptide may belabeled with a detectable moiety. The detectable moiety may be selectedfrom the group consisting of: a fluorescent label, a digoxigenin, abiotin, an enzyme, a radioactive atom, a paramagnetic ion, and achemiluminescent label.

In another embodiment, the agent comprises a nucleic acid molecule whichis a ribozyme or an antisense nucleic acid molecule.

The agent may comprise a peptide having an amino acid sequencecorresponding to the amino acid sequence of a V-domain of a sRAGE linkedto a second peptide, wherein the second peptide may be an albumin, aglobulin or a peptide chosen from a group of peptides, wherein eachpeptide of the group comprises a different length peptide, and whereinthe sequence of each peptide corresponds to any sequence of amino acidstaken from within amino acid number 31 through amino acid number 281 ofthe human, bovine, mouse or rat sRAGE protein.

The abbreviations used herein for amino acids are those abbreviationswhich are conventionally used: A=Ala=Alanine; R=Arg=Arginine;N=Asn=Asparagine; D=Asp=Aspartic acid; C=Cys=Cysteine; Q=Gln=Glutamine;E=Glu=Gutamic acid; G=Gly=Glycine; H=His=Histidine; I=Ile=Isoleucine;L=Leu=Leucine; K=Lys=Lysine; M=Met=Methionine; F=Phe=Phenyalanine;P=Pro=Proline; S=Ser=Serine; T=Thr=Threonine; W=Trp=Tryptophan;Y=Tyr=Tyrosine; V=Val=Valine. The amino acids may be L- or D-aminoacids. An amino acid may be replaced by a synthetic amino acid which isaltered so as to increase the half-life of the peptide or to increasethe potency of the peptide, or to increase the bioavailability of thepeptide.

The peptide or polypeptide of the present invention may comprisealterations to the sequences provided in SEQ ID NOS:1 to 6. The peptideof the present invention may comprise alterations in sequence which donot affect the functionality of the peptide in a negative way, but whichmay increase the functionality of the peptide in a position way, e.g.increase the potency of the peptide. Some examples of such alterationsto the human sequence of the first 30 amino acids (1-30) of the V-domainof sRAGE (SEQ ID NO:1) are listed hereinbelow as examples:

-   -   (a) Substitute D-alanine for L-alanine in position 6;    -   (b) Substitute D-lysine for L-lysine in position 15;    -   (c) Substitute D-alanine for L-alanine in position 6 and        D-lysine for L-lysine in position 15;    -   (d) Omit amino acids 1-5 of the V domain, making the N-amino end        group L-alanine;    -   (e) Omit amino acids 1-5 of the V domain making the N-amino acid        D-alanine;    -   (f) Substitute D-lysine for the L-lysine in the amino acid        number “30” position of the V domain of Sequence I.D. No. 1;    -   (g) Substitute L-arginine for L-lysine in the 30 position of the        V domain;    -   (h) Substitute L-arginine for L-lysine in the 30 position of the        V domain and add glycine as the carboxyl terminal group to        produce a 31 amino acid peptide;    -   (i) Substitute L-arginine for L-lysine in the 30 position of the        amino acid peptide containing the amino acid sequence of 6-30        described for the V domaine of sRAGE;    -   (j) Substitute L-arginine for L-lysine in the 30 position of the        amino acid peptide containing the amino acid sequence of 6-30        described for the V domaine of sRAGE and add glycine as the        carboxyl terminal group to produce a 25 amino acid sequence        peptide;    -   (k) Substitute D-lysine for L-lysine in the 30 position of the        6-30 amino acid sequence designated for the V domain;    -   (l) Substitute D-lysine for L-lysine in the 30 position of the        6-30 amino acid sequence designated for the V domaine and add        L-alanine at the C-terminal position of the new 26 amino acid        peptide;    -   (m) Substitute D-valine for L-valine in the 13 position of the V        domaine 30 amino acid peptide designated 6-30 of the sRAGE V        domain;    -   (n) Substitute D-valine for L-valine in the 13 position of the        25 amino acid peptide designated 6-30 of the sRAGE V domain;    -   (o) Substitute D-alanine for L-alanine in the 6 position of the        30 amino acid peptide and D-valine for L-valine in the 13        position of the 30 amino acid of the V domain;    -   (p) Substitute D-alanine for L-alanine in the 6 position and        D-valine for L-valine in the 13 position of the 25 amino acid        peptide designated 6-30 of the V domain of sRAGE;    -   (q) the above-listed (a)-(p) peptides derivatized through the        carboxylic acid of position 30 with albumin, globulins or        different length peptides composed of amino acids contained        within positions 31 through 281 of the human, mouse, rat or        bovine sRAGE protein.

In addition to naturally-occurring forms of polypeptides derived fromsRAGE, the present invention also embraces other polypeptides such aspolypeptide analogs of sRAGE which have the equivalent funcationality ofthe peptide of SEQ ID NO:1 or 2 or a more potent or more positivefunctionality. Such analogs include fragments of sRAGE. Following theprocedures of the published application by Alton et al. (WO 83/04053),one can readily design and manufacture genes coding for microbialexpression of polypeptides having primary conformations which differfrom that herein specified for in terms of the identity or location ofone or more residues (e.g., substitutions, terminal and intermediateadditions and deletions). Alternately, modifications of cDNA and genomicgenes can be readily accomplished by well-known site-directedmutagenesis techniques and employed to generate analogs and derivativesof sRAGE polypeptide. Such products share at least one of the biologicalproperties of sRAGE but may differ in others. As examples, products ofthe invention include those which are foreshortened by e.g., deletions;or those which are more stable to hydrolysis (and, therefore, may havemore pronounced or longerlasting effects than naturally-occurring); orwhich have been altered to delete or to add one or more potential sitesfor O-glycosylation and/or N-glycosylation or which have one or morecysteine residues deleted or replaced by e.g., alanine or serineresidues and are potentially more easily isolated in active form frommicrobial systems; or which have one or more tyrosine residues replacedby phenylalanine and bind more or less readily to target proteins or toreceptors on target cells. Also comprehended are polypeptide fragmentsduplicating only a part of the continuous amino acid sequence orsecondary conformations within sRAGE, which fragments may possess oneproperty of sRAGE and not others. It is noteworthy that activity is notnecessary for any one or more of the polypeptides of the invention tohave therapeutic utility or utility in other contexts, such as in assaysof sRAGE antagonism. Competitive antagonists may be quite useful in, forexample, cases of overproduction of sRAGE.

Of applicability to polypeptide analogs of the invention are reports ofthe immunological property of synthetic peptides which substantiallyduplicate the amino acid sequence in naturally-occurring proteins,glycoproteins and nucleoproteins. More specifically, relatively lowmolecular weight polypeptides have been shown to participate in immunereactions which are similar in duration and extent to the immunereactions of physiologically-significant proteins such as viralantigens, polypeptide hormones, and the like. Included among the immunereactions of such polypeptides is the provocation of the formation ofspecific antibodies in immunologically-active animals [Lerner et al.,1981; Ross et al., 1981; Walter et al., 1981; Wong et al., 1982; Baronet al., 1982; Dressman et al., 1982; and Lerner, Scientific American,1983. See also, Kaiser et al., 1984] relating to biological andimmunological properties of synthetic peptides which approximately sharesecondary structures of peptide hormones but may not share their primarystructural conformation.

The polypeptide of the present invention may be a peptidomimeticcompound which may be at least partially unnatural. The peptidomimeticcompound may be a small molecule mimic of a portion of the amino acidsequence of sRAGE. The compound may have increased stability, efficacy,potency and bioavailability by virtue of the mimic. Further, thecompound may have decreased toxicity. The peptidomimetic compound mayhave enhanced mucosal intestinal permeability. The compound may besynthetically prepared. The compound of the present invention mayinclude L-, D-, DL- or unnatural amino acids, alpha,alpha-disubstitutedamino acids, N-alkyl amino acids, lactic acid (an isoelectronic analogof alanine). The peptide backbone of the compound may have at least onebond replaced with PSI-[CH═CH] (Kempf et al. 1991). The compound mayfurther include trifluorotyrosine, p-Cl-phenylalanine,p-Br-phenylalanine, poly-L-propargylglycine, poly-D,L-allyl glycine, orpoly-L-allyl glycine.

One embodiment of the present invention is a peptidomimetic compoundhaving the biological activity of preventing accelerated athersclerosisin a subject wherein the compound has a bond, a peptide backbone or anamino acid component replaced with a suitable mimic. Examples ofunnatural amino acids which may be suitable amino acid mimics includeβ-alanine, L-α-amino butyric acid, L-γ-amino butyric acid, L-α-aminoisobutyric acid, L-ε-amino caproic acid, 7-amino heptanoic acid,L-aspartic acid, L-glutamic acid, cysteine (acetamindomethyl),N-ε-Boc-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L-methioninesulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine,N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine,Boc-hydroxyproline, Boc-L-thioproline. (Blondelle, et al. 1994; Pinilla,et al. 1995).

In accordance with the method of this invention, the agent may comprisea peptide, a peptidomimetic, an organic molecule, a carbohydrate, alipid, an antibody or a nucleic acid. The peptide of this invention maycomprise an advanced glycation endproduct peptide or a portion thereof,a receptor for advanced glycation endproduct peptide or a portionthereof, a soluble receptor for advanced glycation endproduct peptide ora portion thereof. The peptide of the present invention may comprise anypart of the first 112 amino acids of the sRAGE protein. The peptide ofthe present invention may comprise the V-domain of a soluble RAGEprotein. The peptide of the present invention may be a smaller portionof the V-domain of a soluble RAGE protein. The peptide of the presentinvention may be a peptide which corresponds to the V-domain of humanRAGE, mouse RAGE, rat RAGE, bovine RAGE or fish RAGE.

In accordance with the method of this invention, the agent may be apeptide (polypeptide), a peptidomimetic, an organic molecule, acarbohydrate, a lipid, an antibody or a nucleic acid. In the case ofpolypeptides, the polypeptide may be an advanced glycation endproduct(AGE) polypeptide or a portion thereof, a receptor for advancedglycation endproduct polypeptide or a portion thereof, a solublereceptor for advanced glycation endproduct polypeptide or a portionthereof, e.g., soluble RAGE, or a recombinant polypeptide. Thepolypeptide may be the V-domain of sRAGE, or amino acids 1-30 of theV-domain of sRAGE. The polypeptide of this invention may comprise anadvanced glycation endproduct polypeptide or a portion thereof, areceptor for advanced glycation endproduct polypeptide or a portionthereof, a soluble receptor for advanced glycation endproductpolypeptide or a portion thereof. The polypeptide of the presentinvention may comprise any part of the first 112 amino acids of thesRAGE protein. The polypeptide of the present invention may comprise theV-domain of a soluble RAGE protein. The polypeptide of the presentinvention may be a smaller portion of the V-domain of a soluble RAGEprotein. The polypeptide of the present invention may be a polypeptidewhich corresponds to the V-domain of human RAGE, mouse RAGE, rat RAGE,bovine RAGE or fish RAGE. The polypeptide may be synthesized chemicallyor produced by standard recombinant DNA methods. In the case ofantibodies, the antibody may be an anti-RAGE antibody or an anti-RAGEF(ab′)₂ fragment.

Of applicability to polypeptide analogs of the invention are reports ofthe immunological property of synthetic peptides which substantiallyduplicate the amino acid sequence extant in naturally-occurringproteins, glycoproteins and nucleoproteins. More specifically,relatively low molecular weight polypeptides have been shown toparticipate in immune reactions which are similar in duration and extentto the immune reactions of physiologically-significant proteins such asviral antigens, polypeptide hormones, and the like. Included among theimmune reactions of such polypeptides is the provocation of theformation of specific antibodies in immunologically-active animals[Lerner et al., Cell, 23, 309-310 (1981); Ross et al., Nature, 294,654-658 (1981); Walter et al., Proc. Natl. Acad. Sci. USA, 78, 4882-4886(1981); Wong et al., Proc. Natl. Sci. USA, 79, 5322-5326 (1982); Baronet al., Cell, 28, 395-404 (1982); Dressman et al., Nature, 295, 185-160(1982); and Lerner, Scientific American, 248, 66-74 (1983). See also,Kaiser et al. [Science, 223, 249-255 (1984)] relating to biological andimmunological properties of synthetic peptides which approximately sharesecondary structures of peptide hormones but may not share their primarystructural conformation.

The present invention also encompasses a pharmaceutical compositionwhich comprises a therapeutically effective amount of the peptide havingan amino acid sequence corresponding to the amino acid sequence of aV-domain of a receptor for advanced glycation endproduct (RAGE) and apharmaceutically acceptable carrier. The carrier may be a diluent, anaerosol, a topical carrier, an aqueous solution, a nonaqueous solutionor a solid carrier. The carrier may be a polymer or a toothpaste. Thepharmaceutical composition may comprise the peptide having an amino acidsequence corresponding to the amino acid sequence of a V-domain of aRAGE linked to a second peptide, wherein the second peptide may be analbumin, a globulin or a peptide chosen from a group of peptides,wherein each peptide of the group comprises a different length peptide,and wherein the sequence of each peptide corresponds to any sequence ofamino acids taken from within amino acid number 31 through amino acidnumber 281 of the human sRAGE protein.

In one embodiment of the invention, the agent consists essentially of aportion of the peptide consisting of an amino acid sequencecorresponding to the amino acid sequence of a V-domain of a receptor foradvanced glycation endproduct. In one embodiment, the agent consists ofsRAGE. In one embodiment, the agent consists of the V-domain of sRAGE.

In one embodiment of the invention, the agent is an inhibitor of theinteraction between RAGE and AGE or RAGE and another binding partner.The inhibitor comprises a peptide, a peptidomimetic compound, a nucleicacid molecule, a small molecule, an organic compound, an inorganiccompound, or an antibody or a fragment thereof. The inhibitor may be theisolated peptide having an amino acid sequence corresponding to theamino acid sequence of a V-domain of a receptor for advanced glycationendproduct. In one embodiment, the inhibitor is capable of specificallybinding to the amyloid-β peptide. In one embodiment, the agent consistsessentially of a peptide having the amino acid sequenceA-Q-N-I-T-A-R-I-G-E-P-L-V-L-K-C-K-G-A-P-K-K-P-P-Q-R-L-E-W-K (SEQ IDNO:7). In another embodiment, the agent consists essentially of apeptide consisting of the amino acid sequence A-Q-N-I-T-A-R-I-G-E (SEQID NO:8).

In one embodiment of the invention, the agent is an antibody. Inaccordance with the method of this invention, the antibody may comprisean anti-RAGE antibody or an anti-RAGE F(ab′)₂ fragment. The fragment ofthe antibody which is useful in the present invention is that whichbinds the antigen. Antibodies may be humanized or chimeric. The antibodymay be a human antibody, a primate antibody, or a murine antibody. Theportion or fragment of the antibody may comprise a complementaritydetermining region or a variable region. In one embodiment, the antibodymay be capable of specifically binding to the receptor for advancedglycation endproduct. The antibody may be a monoclonal antibody, apolyclonal antibody.

The agent may be conjugated to a carrier. The peptide or agent may belinked to an antibody, such as a Fab or a Fc fragment for specificallytargeted delivery. The carrier may be a diluent, an aerosol, a topicalcarrier, an aqeuous solution, a nonaqueous solution or a solid carrier.

As used herein, the term “suitable pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, acetate buffered saline solution(a likely vehicle for parenteral administration), water, emulsions suchas an oil/water emulsion or a triglyceride emulsion, various types ofwetting agents, tablets, coated tablets and capsules. An example of anacceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid®.

When administered orally or topically, such agents and pharmaceuticalcompositions would be delivered using different carriers. Typically suchcarriers contain excipients such as starch, milk, sugar, certain typesof clay, gelatin, stearic acid, talc, vegetable fats or oils, gums,glycols, or other known excipients. Such carriers may also includeflavor and color additives or other ingredients. The specific carrierwould need to be selected based upon the desired method of deliver,e.g., PBS could be used for intravenous or systemic delivery andvegetable fats, creams, salves, ointments or gels may be used fortopical delivery.

This invention also provides for pharmaceutical compositions includingtherapeutically effective amounts of protein compositions and/or agentscapable of inhibiting the binding of an amyloid-β peptide with areceptor for advanced glycation endproduct in the subject of theinvention together with suitable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers useful in treatment of neuronaldegradation due to aging, a learning disability, or a neurologicaldisorder. Such compositions are liquids or lyophilized or otherwisedried formulations and include diluents of various buffer content (e.g.,Tris-HCl., acetate, phosphate), pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, detergents (e.g.,Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents(e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzylalcohol, parabens), bulking substances or tonicity modifiers (e.g.,lactose, mannitol), covalent attachment of polymers such as polyethyleneglycol to the agent, complexation with metal ions, or incorporation ofthe agent into or onto particulate preparations of polymeric agents suchas polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes,micro emulsions, micelles, unilamellar or multi lamellar vesicles,erythrocyte ghosts, or spheroplasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance of the agent or composition. The choice ofcompositions will depend on the physical and chemical properties of theagent capable of alleviating the symptoms of the cognitive disorder ofmemory or the learning disability in the subject.

The agent of the present invention may be delivered locally via acapsule which allows sustained release of the agent or the peptide overa period of time. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines) and the agent coupled toantibodies directed against tissue-specific receptors, ligands orantigens or coupled to ligands of tissue-specific receptors. Otherembodiments of the compositions of the invention incorporate particulateforms protective coatings, protease inhibitors or permeation enhancersfor various routes of administration, including parenteral, pulmonary,nasal and oral.

Portions of the agent of the invention may be “labeled” by associationwith a detectable marker substance (e.g., radiolabeled with ¹²⁵I orbiotinylated) to provide reagents useful in detection and quantificationof compound or its receptor bearing cells or its derivatives in solidtissue and fluid samples such as blood, cerebral spinal fluid or urine.

When administered, agents (such as a peptide comprising the V-domain ofsRAGE) are often cleared rapidly from the circulation and may thereforeelicit relatively short-lived pharmacological activity. Consequently,frequent injections of relatively large doses of bioactive agents may byrequired to sustain therapeutic efficacy. Agents modified by thecovalent attachment of water-soluble polymers such as polyethyleneglycol, copolymers of polyethylene glycol and polypropylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline are known to exhibit substantiallylonger half-lives in blood following intravenous injection than do thecorresponding unmodified agents (Abuchowski et al., 1981; Newmark etal., 1982; and Katre et al., 1987). Such modifications may also increasethe agent's solubility in aqueous solution, eliminate aggregation,enhance the physical and chemical stability of the agent, and greatlyreduce the immunogenicity and reactivity of the agent. As a result, thedesired in vivo biological activity may be achieved by theadministration of such polymer-agent adducts less frequently or in lowerdoses than with the unmodified agent.

Attachment of polyethylene glycol (PEG) to agents is particularly usefulbecause PEG has very low toxicity in mammals (Carpenter et al., 1971).For example, a PEG adduct of adenosine deaminase was approved in theUnited States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicityand antigenicity of heterologous compounds. For example, a PEG adduct ofa human protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The compound of the present invention capable of alleviatingsymptoms of a cognitive disorder of memory or learning may be deliveredin a microencapsulation device so as to reduce or prevent an host immuneresponse against the agent or against cells which may produce thecompound. The agent of the present invention may also be deliveredmicroencapsulated in a membrane, such as a liposome.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

Administration

The administration of the agent may be effected by intralesional,intraperitoneal, intramuscular or intravenous injection; by infusion; ormay involve liposome-mediated delivery; or topical, nasal, oral, anal,ocular or otic delivery. The administration may comprise subcutaneous,vaginal, sublingual, uretheral, transdermal, or intrathecal.

The agent may be delivered hourly, daily, weekly, monthly, yearly (e.g.in a time release form) or as a one time delivery. The delivery may becontinuous delivery for a period of time, e.g. intravenous delivery. Theagent or pharmaceutical composition of the present invention may bedelivered intercranially or into the spinal fluid.

In accordance with the method of this invention, the therapeuticallyeffective amount may comprise a dose of from about 200 ng/day/kg bodyweight to about 200,000 ng/day/kg body weight or from about 50 ng/day/kgto about 500,000 ng/day/kg body weight.

In the practice of the method administration may comprise daily, weekly,monthly or hourly administration, the precise frequency being subject tovarious variables such as age and condition of the subject, amount to beadministered, half-life of the agent in the subject, area of the subjectto which administration is desired and the like.

In connection with the method of this invention, a therapeuticallyeffective amount of may include dosages which take into account the sizeand weight of the subject, the age of the subject, the severity of thesymptom, the surface area of the wound, the efficacy of the agent, themethod of delivery of the agent and the history of the symptoms in thesubject. One of ordinary skill in the art would be readily able todetermine the exact dosages and exact times of administration based uponsuch factors. For example, a therapeutically effective amount may a doseof from about 200 ng/day/kg body weight to about 200,000 ng/day/kg bodyweight. In this regard, it has been shown that 24 microgramsadministered intraperitoneally daily (on days 3-9) to wounded diabeticmice resulted in greatly reduced inflammation. In this regard, the dosemay also be administered as a single dose or as a series of doses over aperiod of time.

The effective amount of the agent may comprise from about 0.000001 mg/kgbody weight to about 100 mg/kg body weight. In one embodiment, theeffective amount may comprise from about 0.001 mg/kg body weight toabout 50 mg/kg body weight. In another embodiment, the effective amountmay range from about 0.01 mg/kg body weight to about 10 mg/kg bodyweight. The actual effective amount will be based upon the size of theagent, the biodegradability of the agent, the bioactivity of the agentand the bioavailability of the agent. The agent may be deliveredtopically in a creme or salve carrier. It may be reapplied as neededbased upon the absorbancy of the carrier to the skin or mucosa or wound.If the agent does not degrade quickly, is bioavailable and highlyactive, a smaller amount will be required to be effective. The effectiveamount will be known to one of skill in the art; it will also bedependent upon the form of the agent, the size of the agent and thebioactivity of the agent. One of skill in the art could routinelyperform empirical activity tests for a agent to determine thebioactivity in bioassays and thus determine the effective amount.

Wound Healing

One example of inflammation in a subject is that which is associatedwith a wound in a subject. One embodiment of treating inflammation in asubject is improving wound healing in a subject. The present inventionprovides a method for improving wound healing in a subject whichcomprises administering to the subject a therapeutically effectiveamount of an agent which inhibits binding of advanced glycationendproducts (AGEs) to a receptor for advanced glycation endproducts(RAGE), over a sufficient period of time in a sufficient amount so as toimprove wound healing in the subject.

The present invention provides a method for alleviating inflammation ina subject which comprises administering a therapeutically effectiveamount of an agent which inhibits binding of advanced glycationendproducts to any receptor for advanced glycation endproducts so as totreat symptoms of inflammation in the subject.

There may be other mechanisms by which soluble RAGE may improveinflammation in a subject. Soluble RAGE may have other effects, such asanti-inflammatory effects that are at least in part, independent ofbinding up AGE's and interfering with their ability to activate cellularRAGE.

The mechanism of reducing inflammation in the subject may be biochemicalin nature or competitive in nature.

As used herein “AGE” means an advanced glycation endproduct; “RAGE”means a receptor for an advanced glycation endproduct; “sRAGE” means asoluble form of a receptor for an advanced glycation endproducts, suchas the extracellular two-thirds of the RAGE polypeptide.

In the practice of the methods of the invention a “therapeuticallyeffective amount” is an amount of an agent which is capable ofinhibiting the binding of AGE to any receptor for advanced glycationendproduct (RAGE). Accordingly, the effective amount will vary with thesubject being treated, as well as with the type of inflammation to betreated. For the purposes of this invention, the methods ofadministration are to include, but are not limited to, administrationcutaneously, subcutaneously, intravenously, parenterally, orally,topically, or by aerosol.

Portions of the agent of the invention may be “labeled” by associationwith a detectable marker substance (e.g., radiolabeled with ¹²⁵I orbiotinylated) to provide reagents useful in detection and quantificationof such agent or its receptor bearing cells or its derivatives in solidtissue and fluid samples such as blood, serum, cerebral spinal fluid orurine.

Compositions

The present invention provides compositions consisting essentially of anagent which reduces inflammation and a carrier. The agent may be aninhibitor of the interaction between RAGE and AGEs. The agent may be aninhibitor of the binding activity of RAGE.

When administered, compounds are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive compounds may by required to sustaintherapeutic efficacy. Compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified compounds(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987).Such modifications may also increase the compound's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound adducts less frequently or in lower doses thanwith the unmodified compound.

Attachment of polyethylene glycol (PEG) to compounds is particularlyuseful because PEG has very low toxicity in mammals (Carpenter et al.,1971). For example, a PEG adduct of adenosine deaminase was approved inthe United States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicityand antigenicity of heterologous compounds. For example, a PEG adduct ofa human protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The compound of the present invention capable of improvingwound healing in a subject may be delivered in a microencapsulationdevice so as to reduce or prevent a host immune response against thecompound or against cells which may produce the compound. The compoundof the present invention may also be delivered microencapsulated in amembrane, such as a liposome.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

The invention also provides a kit which comprises a therapeutic amountof an agent, which agent is capable of inhibiting binding of advancedglycation endproducts to a receptor for advanced glycation endproducts,over a sufficient period of time in a sufficient amount so as to treatchronic symptoms of diabetes in the subject. A kit may include acomposition which includes sRAGE or a portion thereof in a form which ispreviously dose regulated and time regulated so that a subject mayeasily take such therapeutic at home or away from a clinical setting.The kit also includes a means for administering the agent to thesubject.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS Example 1 Treating Wound Healing as an Example ofTreating Inflammation

Improved Wound Healing in Diabetic Mice by Treatment with the SolubleReceptor for Advanced Glycation Endproducts (sRAGE)

Ineffective healing of wounds is a serious problem in diabetes,contributing to increased morbidity (Reynolds, 1985; Galloway andShuman, 1963; and Pearl and Kanat, 1988). The reparative response inwound healing is orchestrated by multiple cellular elements which worktogether in many ways, including infiltration of the lesion byinflammatory effector cells. Subsequent to this, fibroblastic elementstogether with inflammatory effector cells provide antibacterialmechanisms and promote removal of necrotic tissue, as well as layingdown of new connective tissue. A fundamental disorder of glucosemetabolism might perturb these complex and interactive protectiveprocesses. Previous work has suggested that cellular dysfunction indiabetic wound healing involves defective neutrophil function (Bagdadeet al., 1978; Nolan et al., 1978; and Mowat and Baum, 1971), delayedinfiltration of the wound with inflammatory cells (Greenhalgh et al.,1990 and Fahey et al., 1991), decreased production of collagen (Goodsonand Hunt, 1977 and Goodson and Hunt, 1986), and diminished activity ofendogenous growth factors, such as basic fibroblast growth factor(Giardino et al., 1994), which could provide a basis for the slowerformation of granulation tissue and wound closure.

Defective wound healing in diabetes continues to be an important causeof morbidity in the postoperative period, following trauma, and in therepair of cutaneous lesions. Advanced Glycation Endproducts (AGEs) arethe result of nonenzymatic glycation/oxidation of proteins/lipids.Accelerated formation and accumulation of AGEs in tissues of patientswith diabetes has been linked, in certain situations, to the developmentof secondary complications. An important means by which AGEs perturbhomeostatic processes is through their interaction with cellular bindingsites; the best characterized of these is Receptor for AGE or RAGE, animmunoglobulin superfamily molecule expressed by endothelium, monocytes,and smooth muscle cells, as well as mesangial cells and neurons. AGEengagement of RAGE leads to endothelial activation, with expression ofadhesion molecules, enhanced procoagulant properties, and diminishedbarrier function; and perturbation of monocytes, with changes in cellmotility and activation, resulting in expression of proinflammatorycytokines. The interaction of AGEs with RAGE-bearing cells, especiallyendothelium and mononuclear phagocytes, may promote chronic cellularactivation thereby preventing optimal wound healing as reflected byformation of granulation tissue and new connective tissue. The dataherein are consistent with this concept: using a secondary intentionwound model in diabetic mice, wound closure is enhanced followingadministration of soluble(s) RAGE, the extracellular domain of thereceptor. These experiments contribute to a long-term goal and long-feltneed, understanding the contribution of cellular interactions of AGEs inthe pathogenesis of diabetic complications.

Poor wound healing in diabetes is likely to be a manifestation of abasic defect in the host inflammatory-reparative response, in additionto possible underlying vascular insufficiency. Exposure ofmacromolecules to aldose sugars results in nonenzymatic glycation andoxidation (Baynes, 1991; Sell and Monnier, 1989; Ruderman et al., 1992;and Vlassara et al., 1994), initially the reversible early glycationadducts, Schiff bases and Amadori products, form. Following furthercomplex molecular rearrangements, the irreversible AGEs come about. Thelatter comprise a heterogenous group of structures characterized byfluorescence, propensity to form cross-links, generation of reactiveoxygen intermediates (ROIs) and interaction with cellular receptors, thebest characterized of which is Receptor for AGE, or RAGE (Schmidt etal., 1992; Neeper et al., 1992; and Schmidt et al., 1994a). AGEsaccumulated in the tissues in diabetes influence end-organ function bytwo general mechanisms: directly, via effects on tissue architecture,consequent to the formation of cross-links and trapping of plasmaproteins, and indirectly, by interaction with cellular elements, such asendothelial cells (Ecs), mononuclear phagocytes (Mps), central tohomeostasis as well as the host response to pathophysiologicallyrelevant stimuli.

Studies have suggested that the extracellular two-thirds of themolecule, soluble or sRAGE, appeared to be able to inhibit theinteraction of circulating AGEs with cellular surfaces (Schmidt et al.,1994b). For example, binding of radiolabelled AGE albumin, a prototypicligand developed in the laboratory, to cultured endothelial cells orperipheral blood-derived mononuclear phagocytes, was inhibited in thepresence of increasing doses of sRAGE. In vivo, clearance ofradiolabelled AGE albumin from the circulation of a normal mouse afterintravenous injection, was delayed upon treatment with sRAGE.Extrapolation of these findings was attempted to the setting of woundhealing. The goal in these studies was to assess the role of AGE-RAGEinteraction in the setting of the host response to wounding.

In order to assess the contribution of AGE-RAGE interaction to defectivewound healing in diabetes, the wound healing response in diabetic wascompared to normal animals, and to determine if blockade of RAGE wouldameliorate wound closure in diabetes. In these studies, it was foundthat administration of soluble RAGE improved wound healing ingenetically-diabetic mice. These data support the hypothesis that RAGEblockade may represent a feasible target for intervention in diabeticwound healing as well as other complications of diabetes, such as renal,retinal, neurological, cardiovascular, cerebrovascular and peripheralvascular diseases. Diabetic subjects experience increased restenosis andlocal problems after angioplasty which suggests that soluble RAGE may bebeneficial in reducing restenosis after balloon/stent injury.

Materials and Methods

Murine model of diabetes. A genetic model ofinsulin-resistant/hyperglycemic diabetes (db+/db+mice) due to anautosomal recessive trait (chromosome 4) which results in abnormalitiesof glucose metabolism and obesity in homozygote mice was employed.Heterozygote mice (db+/+m) do not develop these abnormalities, and areemployed as controls (Coleman, 1982 and Wyse and Dulin, 1970). Diabeticanimals are hyperglycemic (glucose>400 mg/dl by age 3 months), anddevelop abnormalities similar to human complications, including adefective wound repair. Life expectancy of homozygote mice is 6-8months. Wounding studies began when mice reached 8 weeks of age, as AGEsare present by that time.

Model of wound healing. For analysis of wound healing in diabetes, asecondary intention wound model was employed (Greenhalgh et al., 1990),as it stimulates, in part, the clinical situation following breakdown ofskin in an ulcerated area. A full-thickness 1.5×1.5 cm wound was createdon the back of the mouse which was subsequently covered by TEGADERM(clear, plastic closure). The initial area of the wound was measured byplacing a sterile glass slide over the area, and tracing the edges ofthe wound. The area was then determined by using a computer program (NIHImage 157). Serial measurement of the wound dimensions were made on days3, 5, 7, 10, 14 and 17. This data, consistent with those of previousstudies (Greenhalgh et al., 1990), showed significant delay of woundrepair in the diabetic mouse especially within the first 2-3 weeks aftercreation of the wound. Animals in each group were sacrificed at days 17for analysis. Studies began when mice reached 8-10 weeks of age. Incertain experiments, mice were treated with soluble RAGE (theextracellular two-thirds of the molecule) under the TEGADERM on days 3through 9 after the initial wounding procedure.

Immunohistochemistry for Detection of Advanced Glycation Endproducts.

At the time of the wounding procedure, 1.5×1.5 cm wounds were excised,fixed in formalin (10%) and then processed for immunohistochemistryusing affinity-purified anti-AGE IgG (Miyata et al., 1996).

Results

In order to understand the role of RAGE in diabetic wound healing,1.5×1.5 cm wounds were created on the backs of db+/db+ or db+/m+mice. Itwas first determined that there was no statistically-significantdifference in original wound area among the groups of mice receiving thevarious treatment regimens. When sRAGE (200 ng/day) was administeredunder the TEGADERM daily from days 3 through 9, the wound healingobserved in diabetic mice was significantly enhanced compared withdiabetic mice treated with vehicle (phosphate buffered saline; p<0.05;FIG. 1). Furthermore, the healing observed in diabetic mice treated withsRAGE approximated that observed in control, db+/m+mice treated withvehicle (differences were not statistically significant). (FIG. 1).

Consistent with the hypothesis that these findings were due toreceptor-mediated mechanisms, dose-response studies revealed that therewas no enhancement of diabetic wound healing upon administration ofsRAGE, 2,000 ng/day, compared with a daily dose of 200 ng/day(differences were not significant; FIG. 2). However, consistent with thestudies described herein in diabetic mice, treatment with either 200 or2,000 ng/day sRAGE (administered on days 3 through 9) was significantlysuperior to treatment of these mice with phosphate buffered saline whenthe final wound area was measured on day ten after creation of the wound(FIG. 2). However, at a daily dose of sRAGE of 20 ng/day, there was nosignificant difference in wound healing in the diabetic mice receivingsRAGE versus those diabetic mice receiving vehicle. (FIG. 2).

In order to determine if diabetic wounds were enriched inAGE-immunoreactive material, immunohistochemistry was performed ofdiabetic versus control mice wounds using affinity-purified anti-AGEIgG. These studies demonstrated that there was a significant increase inAGE-reactive material in the wound tissue of the diabetic mice (FIG. 3A)compared with the nondiabetic control animals (FIG. 3B).

Discussion

The results of these studies indicate that in diabetic tissue such aswounds, there is increased deposition/formation of AGEs. Such AGEs, uponinteraction with their cellular receptor RAGE, result in the generationof a sustained inflammatory environment in which healing and quiescenceof the potent effector cells and mediators is markedly delayed. It washypothesized that interference with AGE-RAGE interaction might result inaccelerated healing. In these studies, it was demonstrated that localadministration of soluble RAGE improved diabetic wound healing in adose-dependent manner. The specific mechanisms which underlie theefficacy of administration of sRAGE is important. It is possible thatadministration of sRAGE improves any one of a number of important stepsin physiologic wound healing such as inflammation, angiogenesis and/orformation and deposition of new granulation tissue, specificallycollagen.

Taken together, these data suggest that in an AGE-enriched environmentsuch as that observed in diabetes, interference with AGE-cellular RAGEinteraction might result in amelioration of the chronic complications ofdiabetes. Given that RAGE is expressed in the endothelium and smoothmuscle of the vasculature, in mesangial cells, in certain neural andvascular cells of the retina, and in certain neurons of both the centraland peripheral nervous systems as well as other cells, it is likely thatblockade of cellular RAGE might result in improved diabeticcomplications that might otherwise lead to heart attacks, stroke,peripheral vascular disease, amputation of the extremities, kidneydisease/failure, blindness, impotence and neuropathy. RAGE is found inmonocytes and macrophages and may be present in other cell types whereintherapeutic intervention may also be possible. The present studiessupport the concept that administration of sRAGE (or other forms of RAGEblockade; such as recombinant sRAGE, RAGE-based peptides, anti-RAGE IgGor anti-RAGE F(ab′)₂) might present a novel form of therapeuticintervention in this chronic, debilitating disorder.

REFERENCES FOR EXAMPLE 1

-   Bagdade, J. et al. (1978) Impaired granulocyte adherence. A    reversible defect in host defense in patients with poorly controlled    diabetes. Diabetes 27:677-681.-   Baynes, J. (1991) Role of oxidative stress in development of    complications in diabetes. Diabetes 40:405-412.-   Coleman, D. (1982) Diabetes-obesity syndromes in mice. Diabetes 31    (Suppl.):1-6.-   Fahey, T. et al. (1991) Diabetes impairs the late inflammatory    response to wound healing. Surg. Res. 50:308-313.-   Galloway, J. and Shuman, D. (1963) Diabetes and Surgery. Am. J. Med.    34:177-191.-   Giardino, I. et al. (1994) Nonenzymatic glycosylation in vitro and    in bovine endothelial cells after basic fibroblast growth factor    activity. J. Clin. Invest. 94:110-117.-   Goodson, W. and Hunt T. (1977) Studies of wound healing in    experimental diabetes mellitus. J. Surg. Res. 22:221-227.-   Goodson, W. and Hunt T. (1986) Wound collagen accumulation in obese    hyperglycemic mice. Diabetes 35:491-495.-   Greenhalgh, D. et al. (1990) PDGF and FGF stimulate wound healing in    the genetically diabetic mouse. Am. J. Pathol. 136:1235-1246.-   Mowat, A. and Baum, J. (1971) Chemotaxis of polymorphonuclear    leukocytes from patients with diabetes mellitus. NEJM 284:621-627.-   Neeper, M. et al. (1992) Cloning and expression of RAGE: a cell    surface receptor for AGEs. J. Biol. Chem. 267:14998-15004.-   Nolan, C. et al. (1978) Further characterization of the impaired    bactericidal function of granulocytes in patients with poorly    controlled diabetes. Diabetes 27:889-894.-   Pearl, S, and Kanat, I. (1988) Diabetes and healing: a review of the    literature. J. Foot Surg. 27:268-273.-   Reynolds, C. (1985) Management of the diabetic surgical patient. A    systematic but flexible plan is the key. Postgrad. Med. 77:265-279.-   Ruderman, N. et al. (1992) Glucose and diabetic vascular disease.    FASEB J. 6:2905-2914.-   Schmidt, A-M et al. (1994a) Cellular receptors for AGEs.    Arterioscler. Thromb. 14:1521-1528.-   Schmidt, A-M. et al. (1994b) RAGE has a central role in vessel wall    interactions and gene activation in response to AGESs. PNAS, USA    91:8807-8811.-   Schmidt, A-M et al. (1992) Isolation and characterization of binding    proteins for AGEs from lung tissue which are present on the    endothelial surface. J. Biol. Chem. 267:14987-14997.-   Sell, D. and Monnier, V. (1989) Structure elucidation of senescence    cross-link from human extracellular matrix. J. Biol. Chem.    264:21597-21602.-   Vlassara, H. et al. (1994) Pathogenic effects of AGEs:biochemical,    biologic, and clinical implications for diabetes and aging. Lab.    Invest. 70:138-151.-   Wyse, B. and Dulin, W. (1970) The influence of age and dietary    conditions on diabetes in the Db mouse. Diabetologia 6:268-273.

Example 2 Treating Periodontal Disease as an Example of TreatingInflammation

sRAGE Suppresses Accelerated Periodontal Disease in Diabetic Mice.

A model of accelerated periodontal disease in diabetic mice and theeffects of sRAGE were studied. Efficacy of sRAGE is shown in diabetic(streptozotocin C57BL6/J mice). Administration of soluble RAGE(full-length extracellular form of approximately 40 kDa) inhibitsaccelerated alveolar bone loss, which is the hallmark of periodontaldisease.

Intraperitoneal injection of soluble RAGE suppresses bone loss indiabetic mice (db+/db+).

Administration of Soluble(s) RAGE Suppresses Alveolar Bone Loss in aMurine Model of Accelerated Periodontal Disease in Diabetes.

Diabetes was induced in C57BL6/J mice by administration ofstreptozotocin. Diabetes was defined as two serial measurements of serumglucose ≧300 mg/dl. Alternatively, an equal number of mice were treatedwith vehicle for streptozotocin, phosphate buffered saline. One monthafter induction of diabetes, mice were treated every other day for fourconsecutive days with oral/anal administration of the human periodontalpathogen, Porphyromonas gingivalis (Pg) or vehicle, phosphate-bufferedsaline. Two months later, mice were sacrificed and decapitated. Themandibles were isolated and, under a dissecting microscope (Olympus),and using curved microdissecting forceps (2¾ inches, 0.6 mm wide) and ascalpel with a No. 15C blade, the lingual gingival tissue from theposterior area of each quadrant was dissected. Beginning with ahorizontal sulcular incision at the gingival margin of the posteriorteeth, the gingiva was reflected (full thickness) with the scalpelblade. Vertical release incisions were made and the tissue was removed(separating the tissue with a horizontal incision just below themucogingival junction). Tissue was then placed in formalin (10%) forfurther analysis.

After the above procedures, mandibles were exposed to KOH (2%) for threedays and then mechanically defleshed. The jaws (exposure of the lingualsurfaces of each ½ mandible) were then embedded in lab putty. In orderto remove angulation as a variable, the buccal and lingual cusps of theposterior teeth in the ½ mandible were superimposed during embedding andviewed from the lingual surface prior to photography. The defleshed jawswere photographed using the magnifying dissecting microscope andEktachrome® 160T film (color slides).

These slides, at a magnification of 40×, were then magnified further 4×.Images were then traced onto standard tracing paper. For each mouse, atotal area of the distance between the cemento-enamel junction (CEJ) andalveolar bone crest (BC) for a total of 6 posterior teeth was measuredby scanning the tracing into a Macintosh® computer/scanner and theimages analyzed using the program NIH Image 157® (along with AdobePhotoshop® photography program). Total area (in arbitrary pixel units)is reported for each mouse (6 teeth) as indicated in FIG. 4. Statisticalanalysis was performed using one way analysis of variance. At twomonths, a significant 1.55-fold increase in alveolar bone loss wasobserved in diabetic mice compared with nondiabetic controls (seespecific data below). Similar results were observed in db+/db+ mice(genetically-diabetic/insulin-resistant) one month after infection withPg compared with nondiabetic controls (m+/db+).

In order to test if administration of sRAGE would ameliorate alveolarbone loss in Pg-treated C57BL6/J mice, certain diabetic mice weretreated with sRAGE (MSR; either 35 μg IP/day for two months or 3.5 μgIP/day for two months). Control diabetic mice were treated withequimolar concentrations of mouse serum albumin (70 μg IP/day for twomonths). All mice were treated with Pg. At the end of that time,measurements of alveolar bone loss were made. The results are asfollows:

Alveolar bone loss Condition (CEJ to alveolar BC) (I) Diabetic/albumin6,222 ± 406 pixels (SD) (II) Nondiabetic/albumin 4,018 ± 501 pixels (SD)(III) Diabetic/MSR(35 μg/day) 5,242 ± 463 pixels (SD) (IV)Diabetic/MSR(3.5 μg/day) 6,198 ± 427 pixels (SD)

Many diabetic complications may result from the interaction of AGE'swith RAGE to cause cellular perturbation. AGE acts as a ligand for theV-domain of RAGE to mediate such cellular perturbation. This inventionprovides a method for inhibiting cellular perturbation in a subjectassociated with a diabetic condition which comprises administering tothe subject an amount of an inhibitor of the interation of AGE's withRAGE on the surface of a cell effective to inhibit the interaction andthereby inhibit the cellular perturbation in the subject and treat thediabetic condition.

AGE (advanced glycation endproducts) are a heterogeneous group ofcompounds. A single or specific pathogenic AGE compound (s) are beingidentified. Examples of AGEs include but are not limited to: pentosidine(alone or protein-bound modification); carboxymethyllysine (alone orprotein-bound modification); carboxyethyllysine (alone or protein-boundmodification); pyrallines (alone or protein-bound modification);methylglyoxal (alone or protein-bound modification) and ethylglyoxal(alone or protein-bound modification). One of these AGE's may be apathogenic ligand for a specific cellular perturbation due to aninteraction of the AGE with the V-domain of RAGE. This interaction maybe a critical contributory factor in many complications associated withdiabetes. This invention provides for inhibitors of such an interactionwhich may be administered to subjects with diabetic complications.

Cells which may be acted upon by this binding of AGE's to RAGE on thecell surface include endothelial cells, vascular smooth muscle cells,neuronal cells, macrophages, lymphocytes, retinal vascular cells,retinal neuronal cells, mesangial cells and connective tissue cells andcells associated with connective tissue such as cells associated withgingiva and skin. Cells which may be acted upon by this binding of AGE'sto RAGE are not limited to this list but may include other cells presentin a human body. The present invention provides compounds andcompositions which may be useful in inhibiting this interaction, therebyameliorating the cellular perturbation and ultimately the symptomsassociated with diabetes.

Cellular perturbations in those cells that sRAGE, or other peptides oragents provided for by the present invention include but are not limitedto: oxidant stress, hyperpermeability, enhanced expression of adhesionmolecules such as Vascular Cell Adhesion Moleucle—1; enhanced expressionof tissue factor; enhanced macrophage chemotaxis and activation, such aswith increased production of cytokines and growth factors; enhancedmigration of smooth muscle cells, activation of smooth muscle cells,neuronal oxidant stress and apoptosis. Advanced glycation endproducts(AGE) are the irreversible result of nonenxymatic glycation andoxidation. These AGE's form in the connection with a number ofconditions such as: aging, diabetes, inflammation, renal failure,amyloidoses, and hyperlipidemia. AGE's also form in connection withother disease states and abnormal conditions which are not explicitlylisted herein but which are encompassed by the present invention.

Therapeutic Agents Identified through In Vitro Means, are Shown to beEffective In Vivo for Inhibition of Symptoms Associated with DiabeticComplications.

The therapeutic agent identified may be shown to be effective in woundhealing. In wound healing experiments, the secondary intention woundmodel in genetically diabetic mice would be used. The agent (or peptideor pharmaceutical composition) is applied topically to the wounded area,and wound closure (change in wound area), epithelialization and otherhistologic indices (such as collagen production, extracellular matrixproduction, fibrin, etc.) is measured. Each of these measurements areindices of the effectiveness of the agent on increasing wound healing.

In periodontal disease, genetically diabetic and streptozotocin-treatedmice are utilized as animal model systems to examine bone loss aftertreatment with peptides having the sequence of Seq I.D. No. 1. Bone lossis measured quantitatively via histological methods and geometrical areadeterminations. The peptide of Seq. ID No. 1, V-domain peptide, agent orpharmaceutical composition is administered locally (e.g. “painting on”the agent) and/or systemically. Reduced bone loss is an indication of aneffective agent.

In accelerated atherosclerosis, streptozotocin-treated apoE “knock-out”mice on a normal chow diet are employed as animal models of this diseasecondition. The agent (or peptide or pharmaceutical composition) isadministered systemically, and quantitative data is gathered bymeasuring lesion area in the animals after treatment. This data gives anindication of the effectiveness of each agent. The smaller the lesionarea as compared to non-treated controls, the more effective the agent.

In diabetic impotence, a rat model with streptozotocin-treated animalsis employed in which erections are monitored following administration ofapomorphine. The number and frequency of erections is measured in thepresence and in the absence of the agent and such data is compared so asto evaluate the effectiveness of the agent to inhibit symptoms ofimpotence.

In diabetic retinopathy, diabetic rat and mouse models are used asanimal model systems to measure changes in blood flow and retinalpathology. Again, the agent (or peptide or pharmaceutical composition)is administered systemically, and quantitative data is gathered by bloodflow and qualitative data is gathered by examining retinal pathology inthe animals after treatment.

In diabetic nephropathy, diabetic mice and rat models are employed asanimal models of diabetic nephropathy. Changes in glomerular filtrationrate and renal blood flow are measured in animals given a therapeuticagent and measured in animals given a placebo. In addition, theappearance of protein in the urine and histologic changes in glomeruliare determined in each animal. The effectiveness of the agent isevaluated based upon these measurements in inhibiting diabeticnephropathy.

In diabetic neuropathy, genetically diabetic mice are utilized as ananimal model for the determination of the effectiveness of the agent ofthe present invention. The mice are treated with the compoundsystemically. The mice are then observed to determine changes in nerveconduction velocity and changes in the number of myelinated peripheralnerve fibers. Such data compared with equivalent measurements determinedin an untreated animal will provide an indication of the effectivenessof the agent of the present invention.

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Status of the coronary arteries at necropsy in    diabetes mellitus with onset after age 30 yrs: analysis of 229    diabetic patients with and without clinical evidence of coronary    heart disease and comparison to 183 control subjects. Am. J. Med.    69:498-506, 1980;-   Walter et al., Proc. Natl. Acad. Sci. USA, 78, 4882-4886 (1981);-   Wautier, J. L., C. Zoukourian, O. Chappey, M. P. Wautier, P. J.    Guillausseau, R. Cao, O. Hori, D. Stern and A. M. Schmidt.    Receptor-mediated endothelial cell dysfunction in diabetic    vasculopathy: soluble receptor for advanced glycation endproducts    blocks hyperpermeability. J. Clin. Invest. 97:238-243, 1996;-   Wishik, C. (1989) Curr. Opin. Cell Biol. 1, 115-122;-   Wang et al., Proc. Natl. Sci. USA, 79, 5322-5326 (1982);-   Wu, J., Rogers, L., Stern, D., Schmidt, A. M. and Chiu, D. T. W. The    soluble receptor for Advanced Glycation Endproducts (sRAGE)    ameliorates impaired wound healing in diabetic mice. Abstract    booklet, Plastic Surgery Research Council. Abstract #77, p. 43,    1997;-   Yan, S. D., Schmidt, A. M. Anderson, G., Zhang, J., Brett, J.,    Zou, Y. S., Pinsky, D., and Stern, D. Enhanced cellular oxidant    stress by the interaction of advanced glycation endproducts with    their receptors/binding proteins. J. Biol. Chem. 269:9889-9897,    1994;-   Yan, S D, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, M.    Nagashima, J. Morser, A. Migheli, P. Nawroth, G. Godman, D. Stern    and A. M. Schmidt. RAGE and amyloid-β peptide neurotoxicity in    Alzheimer's disease. Nature 382:685-691, 1996;-   Yan, S D, Zhu, H., Fu, J., Yan, S. F., Roher, A., Tourtellotte, W.,    Rajavashisth, T., Chen, X., Stern, D. and Schmidt, A. M.    Amyloid-beta peptide-RAGE interaction elicits nauronal expression of    M-CSF: a proinflammatory pathway in Alzheimer's disease. Proc. Natl.    Acad. Sci. 94:5296-5301, 1997;-   Yankner, B., et al. (1990) Science 250:279-282, 1990.

Example 3 Treating Delayed Type Hypersensitivity as an Example ofTreating Inflammation

Interaction of EN-RAGE (Extracellular Novel Rage Binding Protein) withReceptor for AGE (RAGE) Perpetuates Inflammatory Responses Suppressionof Delayed-Type Hypersensitivity Reactions with Soluble Receptor for Age(sRAGE)

Expression of RAGE, the Receptor for Advanced Glycation Endproducts, isincreased in the setting of inflammation. Here we report a new member ofthe calgranulin family of proinflammatory cytokines called EN-RAGE (orExtracellular Novel RAGE-binding protein), which interacts with RAGE oncells such as endothelial cells, to alter cellular properties in amanner consistent with perturbation. Administration of soluble RAGE (theextracellular ligand binding domain of RAGE; sRAGE) or anti-RAGE oranti-EN-RAGE F(ab′)₂ fragments markedly attentuated inflammation in amodel of delayed hypersensitivity. These data link RAGE to theinflammatory response and identify EN-RAGE and RAGE as novel targets foranti-inflammatory intervention. Soluble RAGE, furthermore, is thus aprototypic structure for the design of a new class of anti-inflammatoryagents.

The Receptor for AGE (RAGE) is a member of the immunoglobulinsuperfamily of cell-surface molecules (1-2). Originally identified andcharacterized as a cellular receptor for glucose (aldose sugar)-modifiedproteins, or Advanced Glycation Endproducts (AGEs) (3-13), RAGE hassubsequently been reported to interact with other ligands, in bothsettings of normal development and in Alzheimer's disease (14-16). Innormal development, RAGE interacts with amphoterin, a polypeptide whichmediates neurite outgrowth in cultured embryonic neurons. In thosestudies, either anti-RAGE F(ab′)₂ or soluble RAGE (sRAGE) inhibitedneurite outgrowth on amphoterin-coated matrices, but not on matricescoated with other substrates such as laminin or poly-1-lysine (3). Inlater studies, RAGE was identified as a receptor on neurons andmicroglia for amyloid-β-peptide, a polypeptide linked to thepathogenesis of neuronal toxicity and death in Alzheimer's disease.

In unpublished observations from our laboratory, we identified thatincreased RAGE expression was noted in the vascular and inflammatorycells of inflammatory lesions, such as in the kidney tissue frompatients with active lupus nephritis (FIG. 5). We therefore hypothesizedthat RAGE might interact with alternative ligand(s) in that setting inorder to, perhaps, participate in the inflammatory response.

Herein, the findings demonstrate that RAGE interacts with a moleculewith close homology to calgranulin C. We have termed this molecule,EN-RAGE (Extracellular Novel RAGE binding protein) and show thatEN-RAGE:RAGE interaction activates cells such as endothelial cells whichare importantly involved in the inflammatory response. In a model ofmurine delayed hypersensitivity, administration of soluble RAGE (sRAGE),which contains the ligand interaction domain, inhibits the developmentof cellular activation and inflammation. These findings identify RAGE asa new target for anti-inflammatory intervention.

Materials and Methods Isolation and Purification of EN-RAGE.

Bovine lung acetone powder (SIGMA®) was subjected to solubilization inbuffer containing tris (0.02M, pH 7.4); NaCl (0.15M); octyl-β-glucoside(1%); and protease inhibitors (PMSF and aprotinin). After serialchromatography onto SP sepharose (Pharmacia LKB®), and affi-gel 10 resin(BIO-RAD®) to which had been adsorbed purified soluble human RAGE(prepared from a baculovirus expression system), RAGE-binding proteinswere identified based on a screening assay employing immobilized columnfraction (Nunc Maxisorp dishes) (NUNC®) and ¹²⁵-I-labelled sRAGE asabove. After elution with heparin-containing buffer (1 mg/ml), positivefractions were identified. RAGE-binding proteins were subjected tosequence analysis.

Cloning of EN-RAGE. The cDNA for EN-RAGE was cloned from a bovine lunglibrary and placed into a baculovirus expression system. In this system,EN-RAGE, which lacks a leader sequence, was synthesized within Sf9cells. EN-RAGE was then purified after solubilization of the cells indetergent-containing buffer, and sequential purification onhydroxylapatite and heparin-containing resins. The final productdisplayed a single band on Coomassie-stained SDS-PAGE gels and wasdevoid of endotoxin after chromatography onto Detoxi-gel columns(PIERCE®). Absence of detectable endotoxin was confirmed using limulusamebocyte assay (SIGMA®).

Sequence analysis. After SDS-PAGE identified an ≈12 kDa polypeptide withRAGE-binding activity, the gel band was eluted according topreviously-published methods (17). The published method was modified byaddition of a final wash of two aliquots (0.1 ml each) of guanidine(5.0M), urea (5.0M), trifluoroacetic acid (0.2-0.2%), acetonitrile(10%), and Zwittergent 3-08 (1.0%) (Calbiochem) to ensure that proteinwas completely washed from the filter. Amino-terminal sequence analysiswas performed. Automated Edman degradation was carried out employing anHP-G1005A sequencer (Hewlett Packard Analytical Instruments). In orderto obtain internal sequence, the gel bands were treated as above forelution, except that the extraction buffer contained half the usualamount of SDS (1). Endoproteinase Lys-C (1 μg) (Boehringer Mannheim) wasadded and the sample incubated overnight. The digest was thenfractionated by microbore HPLC (Michrom Bioresources) on a 1 mm×50 mmPLRP-S column (Polymer Laboratories, Ltd.). The gradient utilized was 2%per minute from acetonitrile (5-75%) in trifluoroacetic acid (0.1%) andfractions were collected at 30 second intervals. Absorbance wasmonitored at 214 nm and fractions that corresponded to chromatographicpeaks were then subjected to sequence analysis.

Endothelial cell activation. Human umbilical vein endothelial cells wereisolated, characterized and maintained as previously described (18).Cells were cultured in serum-free RPMI 1640 without endothelial cellgrowth factor for 24 hrs and then stimulated with the indicatedconcentrations of EN-RAGE. Where indicated, cells were pretreated withrabbit anti-human RAGE IgG, nonimmune rabbit IgG; in certain cases,EN-RAGE was pretreated with the indicated concentration of soluble RAGE(sRAGE) for 2 hrs prior to stimulation with EN-RAGE. After eight hrsstimulation with EN-RAGE, cells were fixed with paraformaldehyde (2%)for 30 mins, washed twice with PBS, treated with PBS containing non-fatdry milk (5%) and BSA (2.5%) to block nonspecific binding sites on thecell surface. Cell surface ELISA employing anti-VCAM-1 IgG (Santa CruzBiotechnologies, Santa Cruz, Calif.) was performed. Assessment offunctional VCAM-1 activity was determined using ⁵¹Cr-labelled Molt-4cells (ATCC) as previously described (10).

Delayed hypersensitivity model. A murine model of delayedhypersensitivity was established based on previously-published studies(19). Female CF-1 mice (Charles River laboratories), 6 weeks of age,were sensitized by subcutaneous injection over the left inguinal lymphnode of an emulsion (0.1 ml) containing methylated BSA (mBSA; 25 mg/ml;SIGMA®), NaCl (0.9%), dextran (5-40×10⁶ MW; 50 mg/ml; SIGMA®) andFreund's incomplete adjuvant (50%; ICN Biomedical). Three weeks later,the left plantar hind paw was injected subcutaneously with mBSA (0.4mg/ml; 0.050 ml). Where indicated, mice were pretreated byintraperitoneal injection with sRAGE (indicated dose), mouse serumalbumin (SIGMA®), immune or nonimmune F(ab′)₂ fragments (prepared usinga kit from Pierce) 24 and 12 hrs prior to, and 6 and 12 hrs after localchallenge with mBSA. 24 hrs after injection of foot pad with mBSA,clinical score of foot pad was performed; mice were then humanelysacrificed and feet fixed in formalin (10%) or frozen for furtheranalysis. Histologic score was performed on sections of foot stainedwith hematoxylin and eosin (SIGMA®). The clinical score was defined asfollows (scale; 1-5): 1=no inflammation and thus identical to untreatedfoot; 2=slight rubor and edema; 3=severe rubor and edema with wrinklingof the skin of the foot pad; 4=severe rubor and edema without wrinklingof the skin of the foot pad; and 5=severe rubor and edema resulting inspreading of the toes. The histologic score after hematoxylin and eosinstaining was defined as follows (scale; 1-5): 1=no leukocyticinfiltration with slight subcutaneous edema; 2=slight perivascularleukocytic infiltration with slight subcutaneous edema; 3=severeleukocytic infiltration without granulomata; and 4=severe leukocyticinfiltration with granulomata.

Results

Identification of EN-RAGE. After a serial series of experiments designedto identify RAGE-binding proteins from bovine lung extract (from whereRAGE was originally purified), an ≈12 kDa polypeptide was identified.Upon sequence analysis, this polypeptide was found to bear significanthomology to members of the calgranulin C family of proteins (Table 1)(20-21). This class of proteins exist intracellularly withininflammatory cells. Upon release in inflamed loci, we postulated theymight be able to, in turn, engage and activate other cells alreadyrecruited into the inflammatory response. Thus, this might represent animportant means by which the inflammatory response might be propagatedand sustained, thereby increasing the probability of cellular injury.

EN-RAGE activates endothelial cells in a RAGE-dependent manner. To testthis hypothesis, EN-RAGE was purified as described above and incubatedwith endothelial cells. Incubation of EN-RAGE with HUVEC resulted inincreased cell surface Vascular Cell Adhesion Molecule-1 (VCAM-1) in aRAGE-dependent manner (FIG. 6). These data suggested that in aninflammatory focus, interaction of EN-RAGE with EC RAGE might representa means by which to further propagate an inflammatory response.Consistent with increased VCAM-1 antigen on the surface ofEN-RAGE-treated ECs, increased binding for Molt-4 cells (which bear theligand for VCAM-1, VLA-4), ensued (FIG. 7). While incubation with eitherBSA or non-immune IgG did not affect the ability of EN-RAGE to activateEC VCAM-1, incubation with either sRAGE or anti-RAGE F(ab′)₂significantly attenuated the ability of EN-RAGE to increase Molt-4binding to treated HUVEC.

We sought to test these hypotheses in in vivo models. We demonstratedthat in diabetic mice, in which the ligand for RAGE is likely to be, atleast in part, products of glycation/oxidation of proteins/lipids, theAdvanced Glycation Endproducts, or AGEs, administration of the soluble,ligand-binding portion of RAGE (soluble or sRAGE), suppressedaccelerated atherosclerosis in diabetic apolipoprotein E null mice (12)and improved wound healing in genetically-diabetic db+/db+ mice (22).Thus, the biologic effects of EN-RAGE in highly-inflammatory foci, suchas those characterized by models of granulomatous inflammatory lesions(delayed hypersensitivity), could be suppressed in the presence ofsRAGE.

To test this, we studied a model of delayed hypersensitivity (DH) inwhich mice were first sensitized by injection of methylated BSA (mBSA;which does not bind RAGE) over the inguinal lymph nodes of female CF-1mice. Three weeks after sensitization, mice were challenged with mBSA byinjection into the hind foot pad. An inflammation score was designed ona scale of 1-9 which included both clinical score (1-4) and histologicscore (1-5) as indicated in FIG. 8.

Consistent with our hypothesis, administration of sRAGE suppressedinflammation upon injection of mBSA into the foot pad of micepreviously-sensitized with mBSA over the lymph nodes, in adose-dependent manner (FIG. 8). At a dose of 100 μg sRAGE, inflammationwas markedly suppressed (p<0.01). In contrast, administration of mouseserum albumin, had no effect on the appearance of the inflammatorylesion (FIG. 8). Consistent with an important role for EN-RAGE and RAGEin the development of inflammation in this model, treatment of the micewith either anti-EN-RAGE F(ab′)₂ or anti-RAGE F(ab′)₂ considerablysuppressed inflammation (p<0.05 in each case compared with treatmentwith nonimmune F(ab′)₂. When mice were treated with both anti-EN-RAGEand anti-RAGE F(ab′)₂, even further suppression of the inflammatoryresponse eventuated (p<0.05 compared with treatment with nonimmuneF(ab′)₂ (FIG. 8).

Discussion

The inflammation phenotype observed in delayed-type hypersensitivityreactions certainly represent the culmination of a complex interplay andcontribution of multiple cell types and their cellular mediators. In thedevelopment of inflammation, an important source of the stimuli may befrom the inflammatory cells themselves. Upon initial recruitment into aninflammatory locus, cells such as neutrophils and macrophages mayrelease mediators such as those of the calgranulin family, includingEN-RAGE, and propagate and sustain the inflammatory response. Suchmediators, such as EN-RAGE, likely require cellular receptors toinitiate events that will culminate in altered gene expression.

Our data strongly suggest that EN-RAGE-RAGE interaction is an importantfactor in these processes. Nearly complete suppression of inflammationwas noted in the presence of sRAGE, in a dose-dependent manner. Basedupon our studies, sRAGE may act as a decoy in this setting to bindEN-RAGE prior to its ability to engage RAGE-bearing cells implicated inthe inflammatory response. Furthermore, in the presence ofanti-RAGE/anti-EN-RAGE or anti-RAGE+anti-EN-RAGE F(ab′)₂, substantialsuppression of inflammation was observed, further indicating a role ofthese factors in the modulation of the inflammatory response.

It is important to note, of course, that alternate mechanisms underlyingthe beneficial effects of sRAGE may be operative in these settings.However, the studies noted above employing the indicated F(ab′)₂fragments, strongly implicate EN-RAGE and RAGE in the evolution of theinflammatory response in this setting.

In conclusion, the studies presented herein implicate RAGE centrally inthe inflammatory response and identify soluble RAGE as a prototypicstructure for the development of novel, anti-inflammatory agents.

Note: FIG. 9 shows the nucleic acid sequence (cDNA sequence) of bovineEN-RAGE.

TABLE 1 Sequence analysis of EN-RAGE and comparison with relatedproteins.   1      10       20       30 EN-RAGE TKLEDHLEGIINIGHQYSVRVGHFDTLNKY N-TERM Endo Lys C B-COAg TKLEDHLEGIINIFHQYSVRVGHFDTLNKR B-CAAFI  TKLEDHLEGIINIFHQYSVRVGHFDTLNKR31       40         50        60 EN-RAGE  ELKQLGTKELPKTLQNXKDQ N-TERMEndo Lys C B-COAg  ELKQLITKELPKTLQNTKDQPTIDKIFQDL B-CAAFI ELKQLITKELPKTLQNTKDQPTIDKIFQDL 61      70       80       90 EN-RAGEN-TERM Endo Lys C      DGAVSFEEFVVLVSRVLK B-COAg DADKDGAVSFEEFVVLVSRVLKTAHIDIHK B-CAAFI  DADKDGAVSFEEFVVLVSRVLKTAHIDIHK(SEQ ID NOS:9, 10, 11, 12, respectively).

EN-RAGE (Extracellular Novel-RAGE Binding Protein) Activated EndothelialCells to Mediate Inflammatory Responses.

The expression of Receptor for AGE (RAGE) is enhanced in inflammatorysettings such as atherosclerosis and autoimmune vasculitities. Wehypothesized that Receptor for AGE (RAGE) might interact withalternative ligands beyond Advanced Glycation Endproducts (AGEs) in suchsettings. We isolated and purified an ≈12 kDa polypeptide from extractof bovine lung which bore homology to the calgranulin family ofproinflammatory mediators. This polypeptide, called EN-RAGE, bindsimmobilized RAGE and endothelial (EC)/macrophage (MP) RAGE in culturewells with Kd≈75 nM, processes blocked in the presence of anti-RAGE IgGor soluble (sRAGE; the extracellular two-thirds of RAGE). In vitro,exposure of cultured ECs to EN-RAGE increased activation of NF-kB,expression of cell-surface VCAM-1 (4.3-fold compared to treatment withbovine serum albumin BSA), and adhesion of Molt-4 cells (which bearVLA-4, the counter-ligand for VCAM-1) (7-fold compared with BSA), all ina manner inhibited in the presence of anti-RAGE IgG or sRAGE. Exposureof macrophages to EN-RAGE resulted in increased chemotaxis in aRAGE-dependent manner. To test these concepts in vivo, we utilized amodel of delayed hypersensitivity in mice in which footpad injections ofmethylated BSA (mBSA) induce localized inflammation. Pre-treatment(intraperitoneal; IP) with sRAGE prevented mBSA-mediated inflammation ina dose-dependent manner. At 100 μg IP sRAGE, the mBSA-treated footmanifested no inflammation and markedly diminished activation of NF-kBcompared with mice treated with vehicle, mouse serum albumin (MSA);further, elaboration of TNF-alpha into the serum was completelyprevented. Partial anti-inflammatory responses were observed upontreatment of the mice with either anti-RAGE or anti-EN-RAGE F(ab′)2.Nonimmune F(ab′)2 was without effect. Taken together, these findingsindicate that ligands alternative to AGEs such as EN-RAGE activate ECsand MPs, thereby linking RAGE to the generalized inflammatory response.

sRAGE Results in Diminished Mortality After Endotoxemia: A PotentialTreatment for Septic Shock

The use of sRAGE or compounds which are capable of inhibiting theinteraction of EN-RAGE and RAGE could be useful agents for the treatmentof septic shock or sepsis in subjects. It has been shown that a subjectgiven lethal doses of LPS has reduced mortality when the LPS is given inthe presence of sRAGE.

sRAGE and Endotoxemia

Soluble Receptor for AGE (sRAGE) has been shown to prevent inflammationin a model of delayed-type hypersensitivity. Unlike certainanti-inflammatory-type agents, it was believed that sRAGE might exertbeneficial effects when administered in the setting of endotoxemia, aprototypic result of, for example, profound gram negative bacteremia.

When uniformly lethal doses of LPS were administered to Balb/C mice(=750 μg), administration of sRAGE (pre or post LPS injection) preventeddeath in ≈50% of the mice in pilot studies.

These data underscore the proposition that the potent anti-inflammatoryeffects of sRAGE are not associated with an untoward inclination towardmorbidity/mortality due to the presence of septicemia/endotoxemia.sRAGE, therefore, may be a selective anti-inflammatory agent withselective protective effects against maladaptive inflammatory responses.

REFERENCES FOR EXAMPLE 3

-   1. Schmidt, A. M., Vianna, M., Gerlach, M., Brett, J., Ryan, J.,    Kao, J., Esposito, C., Hegarty, H., Hurley, W., Clauss, M., Wang,    F., Pan, Y. C., Tsang, T. C., and Stern, D. Isolation and    characterization of binding proteins for advanced glycosylation    endproducts from lung tissue which are present on the endothelial    cell surface. J. Biol. Chem. 267:14987-14997, 1992.-   2. Neeper, M., Schmidt, A. M., Brett, J., Yan, S. D., Wang, F.,    Pan, Y. C., Elliston, K., Stern, D., and Shaw, A. Cloning and    expression of RAGE: a cell surface receptor for advanced    glycosylation end products of proteins. J. Biol. Chem. 267:    14998-15004, 1992.-   3. Schmidt, A-M, Hori, O, Brett, J, Yan, S-D, Wautier, J-L, and    Stern D. Cellular receptors for advanced glycation end products.    Arterioscler. Thromb. 14:1521-1528, 1994.-   4. Schmidt, A. M., S D Yan, and D. Stern. The Dark Side of Glucose    (News and Views). Nature Medicine 1:1002-1004, 1995.-   5. Yan, S-D, Schmidt, A-M, Anderson, G, Zhang, J, Brett, J, Zou,    Y-S, Pinsky, D, and Stern, D. Enhanced cellular oxidant stress by    the interaction of advanced glycation endproducts with their    receptors/binding proteins. J. Biol. Chem. 269:9889-9897, 1994.-   6. Schmidt, A-M, Yan, S-D, Brett, J, Mora, R, Nowygrod, R, and    Stern D. Regulation of mononuclear phagocyte migration by cell    surface binding proteins for advanced glycosylation endproducts. J.    Clin. Invest. 92:2155-2168, 1993.-   7. Wautier, J L, Chappey, O, Wautier, M P, Hori, O, Stern, D, and    Schmidt A M. Receptor-mediated endothelial dysfunction in diabetic    vasculopathy: sRAGE blocks hyperpermeability. J. Clin. Invest.    97:238-243, 1996.-   8. Miyata, T., Hori, O, Zhang, J H, Yan, S D, Ferran, L, Iida, Y,    and Schmidt, A M. The Receptor for Advanced Glycation Endproducts    (RAGE) mediates the interaction of AGE-b²-Microglobulin with human    mononuclear phagocytes via an oxidant-sensitive pathway:    implications for the pathogenesis of dialysis-related    amyloidosis. J. Clin. Invest. 98:1088-1094, 1996.-   9. Schmidt, A-M, Hasu, M, Popov, D, Zhang, J-H, Chen, J, Yan, S-D,    Brett, J, Cao, R, Kuwabara, K, Gabriela, C, Simionescu, N,    Simionescu, M, and Stern D. Receptor for advanced glycation    endproducts (AGEs) has a central role in vessel wall interactions    and gene activation in response to circulating AGE proteins.    PNAS(USA) 91:8807-8811, 1994.-   10. Schmidt, A M, Hori, O, Chen, J, Brett, J, and Stern, D. AGE    interaction with their endothelial receptor induce expression of    VCAM-1: a potential mechanism for the accelerated vasculopathy of    diabetes. J. Clin. Invest. 96:1395-1403, 1995.-   11. Lander, H. L., Tauras, J. M., Ogiste, J. S., Moss, R. A.,    and A. M. Schmidt. Activation of the Receptor for Advanced Glycation    Endproducts triggers a MAP Kinase pathway regulated by oxidant    stress. J. Biol. Chem. 272:17810-17814, 1997.-   12. Park, L., Raman, K. G., Lee, K. J., Yan, L., Ferran, L. J.,    Chow, W. S., Stern, D., and Schmidt, A. M. Suppression of    accelerated diabetic atherosclerosis by soluble Receptor for AGE    (sRAGE). Nature Medicine 4:1025-1031, 1998.-   13. Wautier J L, Chappey O, Wautier M P, Boval B, Stern D and A M    Schmidt. Interaction of diabetic erythrocytes bearing advanced    glycation endproducts with the endothelial receptor RAGE induces    generation of reactive oxygen intermediates and cellular    dysfunction. Circ. 94 (8):#4139, 1996.-   14. Hori, O., J. Brett, T. Slattery, R. Cao, J. Zhang, J. Chen, M.    Nagashima, D. Nitecki, J. Morser, D. Stern, A. M. Schmidt. The    Receptor for Advanced Glycation Endproducts (RAGE) is a cellular    binding site for amphoterin: mediation of neurite outgrowth and    co-expression of RAGE and amphoterin in the developing nervous    system. J. Biol. Chem. 270:25752-25761, 1995.-   15. Yan, S D, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T.    Slattery, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, G.    Godman, D. Stern, and A. M. Schmidt. RAGE and amyloid-b peptide    neurotoxicity in Alzheimer's disease. Nature 382:685-691, 1996.-   16. Yan, S-D., Zhu, H., Fu, J., Yan, S-F., Roher, A., Tourtellotte,    W., Rajavashisth, T., Chen, X., Stern, D. and Schmidt, A-M.    Amyloid-beta peptide-RAGE interaction elicits neuronal expression of    M-CSF: a proinflammatory pathway in Alzheimer's disease. Proc. Natl.    Acad. Sci. 94:5296-5301, 1997.-   17. Slattery, T. K. and Harkins, R. N. Techniques in protein    chemistry IV, ed. Angeletti, R. H., Academic Press, San Diego,    Calif., 1992.-   18. Jaffe, E., Nachman, R., Becker, C., and Minick, R. Culture of    human endothelial cells derived from umbilical veins. Identification    by morphologic and immunologic criteria. J. Clin. Invest.    52:2745-2756, 1973.-   19. Dunn, C. J., Galinet, L. A., Wu, H., Nugent, R. A.,    Schlachter, S. T., Staite, N. D., Aspar, D. G., Elliott, G. A.,    Essani, N. A., Rohloff, N. A., and Smith, R. J. Demonstration of    novel anti-arthritic and anti-inflammatory effects of    diphosphonates. J. Pharmacology and Experimental Therapeutics 266:    1691-1698, 1993.-   20. Wicki, R., Marenholz, I., Mischke, D., Schafer, B. W., and    Heizmann, C. W. Characterization of the human S100A12 (calgranulin    C, p6, CAAF1, CGRP) gene, a new member of the S100 gene cluster on    chromosome 1q21. Cell Calcium 20:459-464, 1996.-   21. Dell'Angelica, E. C., Schleicher, C. H., and Santome, J. A.    Primary structure and binding properties of calgranulin C, a novel    S100-like calcium-binding protein from pig granulocytes. J. Biol.    Chem. 269:28929-28936, 1994.-   22. Wu J, Rogers L, Stern D, Schmidt A M and Chiu D T W. The soluble    receptor for Advanced Glycation Endproducts (sRAGE) ameliorates    impaired wound healing in diabetic mice. Plastic Surgery Research    Council, Abstract #77, p. 43, 1997.

Example 4 Treatment of Collagen-Induced Arthritis as an Example ofTreating Inflammation Rage (G82S) and Rheumatoid Arthritis: IncreasedSusceptibility and Upregulation of the Inflammatory Response

Receptor for Advanced Glycation Endproducts (RAGE) and itsproinflammatory S100/calgranulin ligands (1) are enriched in joints ofpatients with rheumatoid arthritis (RA) (2-4). Linkage disequilibriumwith an RA-associated HLA-DR4 haplotype (5-6), and a polymorphism of theRAGE gene (G82S) (7-9), suggested a role for RAGE in RA. We demonstratehere that the prevalence of RAGE (G82S) is significantly increased in RAcompared with controls, even in DR4-negative subjects. Cells bearingmutant RAGE (82S), either stably-transfected CHO cells orpatient-derived mononuclear phagocytes, display enhanced responsesfollowing engagement of a prototypic S100/calgranulin, compared withcells expressing wild-type RAGE. Blockade of RAGE in a collagen-inducedarthritis model (10-12) suppressed clinical and histologic evidence ofarthritis, in parallel with diminished levels of proinflammatorymediators and markers of tissue degradation. These findings associateRAGE (G82S) with increased susceptibility to RA, and suggest that RAGE(G82S) primes joint tissue for enhanced inflammation and destruction inevolving arthritis.

MHC-linked genes are widely accepted contributors to susceptibility inautoimmune/inflammatory disorders, though the identity of these geneshas yet to be fully elucidated. Indeed, it is highly likely thatmultiple genes within the MHC, and, perhaps, outside this complex, areinvolved in human autoimmunity. Specifically, in rheumatoid arthritis(RA), polymorphisms at the HLA-DRB1 locus, particularly within theDRB1*04 and DRB1*01 groups of alleles, have been most stronglyassociated with development of disease (5-6, 13). However, despiteintense investigation, a definitive understanding of the link betweenthese alleles and their involvement in susceptibility and/or evolutionof proinflammatory phenomena in RA has not been achieved. The geneencoding RAGE, a multi-ligand member of the immunoglobulin superfamilyof cell surface molecules, is located approximately 400 kb from HLA-DRB1and 300 kb from HLA-DRA, near the junction of MHC Class II and Class III(7). Furthermore, expression of RAGE and its proinflammatory ligands ofthe S100/calgranulin family (termed EN-RAGEs or Extracellular,Newly-identified RAGE binding proteins) (1) is enhanced in affectedrheumatoid synovial tissue (2-4). These considerations led us to testwhether the RAGE polymorphism (G82S), predictably increasinghydrophilicity of a critical portion of the receptor's extracellulardomain involved in ligand binding, might confer increased susceptibilityto RA, as well as enhanced generation of proinflammatory and tissuedestructive mediators important in the evolution of arthritis.

Genomic DNA from RA patients and controls was analyzed using PCRamplification of RAGE exon 3 followed by digestion of the product withAlu I in order to identify subjects bearing the GG, GS and SS genotypes(FIG. 10). Among Caucasian subjects, 76/345 (22a) of patients with RAcarried the S allele, compared with 10/190 (5.3%) control subjects, thusyielding a highly-significant association of RAGE (G82S) with RA, withan estimated relative risk (RR) of 5.0 (95% confidence interval [CI]2.5-10.0), and p<0.001 (Table 2). Since the (G82S) polymorphism hasrecently been found to be in linkage disequilibrium with a commonRA-associated haplotype (9), DRB*0401—DQA1*0301—DQB1*0301, it isdifficult to definitively establish whether RAGE itself contributes todisease risk in the context of this haplotype. Therefore, we focussed onthe subset of patients and controls who do not carry HLA-DR4 haplotypes.As shown in Table 2, when only DR4 negative subjects are considered, theRAGE (G82S) allele continues to exhibit a significant association withRA, which an estimated RR=5.9, (95% CI 1.3-28), and p=0.011. Theseobservations indicate that presence of the RAGE G82S allele confersenhanced susceptibility to RA, even in DR4 negative subjects.Interestingly, no evidence of linkage disequilibrium between RAGE (G82S)and DRB1*0101 was seen.

RA joints display high levels of S100/calgranulins, a family ofpolypeptides associated with inflammatory processes, which transducetheir signal of cellular activation via RAGE. To move from geneticassociations to changes in cell function associated with inflammatorypathways underlying RA, we investigated whether RAGE (G82S) woulddisplay altered affinity and activation of signal transduction pathways,compared with the wild-type receptor, when exposed to a prototypicS100/calgranulin that we previously termed EN-RAGE (1). Chinese hamsterovary (CHO) cells provide a convenient model, as they are devoid ofdetectable RAGE prior to, or after stable transfection with pcDNA3.1vector alone (FIG. 11A). Stably-transfected CHO cells were made withpcDNA3.1 containing either wild-type RAGE or mutant RAGE (82S) (FIG.11A). Radioligand binding studies showed dose-dependent binding of¹²⁵I-EN-RAGE to CHO cells expressing wild-type (≈122±31 nM) and mutantreceptor (Kd≈77±21 nM), though the affinity of binding was greater withthe mutant receptor (FIG. 2 b; p=0.008). In contrast, CHO cells stablytransfected with the empty vector (mock) displayed no specific bindingof ¹²⁵I-EN-RAGE (FIG. 11B). That the interaction of RAGE-bearing CHOcells with ¹²⁵I-EN-RAGE was specific for interaction with RAGE was shownby inhibition of specific binding in the presence of an excess ofsoluble extracellular domain of the receptor (sRAGE), or anti-RAGE IgG(FIG. 1C). In contrast, addition of bovine serum albumin (BSA) ornonimmune IgG was without effect.

These observations led us to test the concept that engagement of RAGE(82S) on transfected CHO cells by EN-RAGE might amplify cellularactivation beyond that seen in cells bearing wild-type RAGE. Incubationof mock-transfected CHO cells with EN-RAGE (10 μg/ml) did not increaseintensity of the bands corresponding to phosphorylated p44/42 MAPkinases (14-15) (FIG. 1D, lanes 1-5). However, exposure of wild-typeRAGE CHO transfectants to EN-RAGE increased by ≈2-fold phosphorylatedp44/42 MAP kinases, compared with cultures incubated with BSA alone(FIG. 11D, lanes 7&6, respectively; p<0.01). CHO transfectants bearingRAGE (82S) incubated with EN-RAGE displayed ≈3.9-fold increase inphosphorylated p44/42 compared with BSA (FIG. 1D, lanes 12 & 11,respectively; p<0.01). Compared with cells expressing wild-type RAGE,CHO cells expressing mutant RAGE displayed significantly increasedphosphorylation of p44/p42 MAP kinases (FIG. 1D, lanes 12 & 7,respectively; p<0.05). In both wild-type RAGE- and mutantRAGE-transfected cells, cellular activation by EN-RAGE was due toengagement of RAGE as demonstrated by suppression of phosphorylation ofp44/p42 in the presence of excess sRAGE (FIG. 11D, lanes 8 & 13,respectively), or anti-RAGE IgG (FIG. 11D, lanes 9&14, respectively).Nonimmune IgG was without effect (FIG. 11D, lanes 10 & 15,respectively). In all cases, levels of nonphosphorylated p44/p42 MAPkinases were identical.

To further support the hypothesis that the presence of mutant RAGE (82S)enhanced activation of key proinflammatory signal transduction pathways,we assessed nuclear translocation of NF-kB in CHO transfectants exposedto EN-RAGE. Electrophoretic mobility shift assays (EMSA) using³²P-labelled consensus NF-κB probe and nuclear extracts frommock-transfected CHO cells showed no increase in intensity of the gelshift band after cultures were exposed to EN-RAGE (FIG. 11E, lane 2).When these experiments were repeated with CHO transfectants expressingwild-type RAGE, there was a prominent ≈5.4-fold increase in intensity innuclear binding activity following incubation of cultures with EN-RAGEcompared to BSA (FIG. 11E, lanes 7&6, respectively). NF-κB activationwas even more striking when RAGE (82S) was substituted for wild-typeRAGE; RAGE (82S) CHO transfectants displayed ≈11.3-fold increasedintensity of the gel shift band consequent to the presence of EN-RAGE,compared to incubation with BSA (FIG. 11E, lanes 12&11, respectively).Thus, RAGE-mediated NF-κB activation due to EN-RAGE was enhanced by≈2.1-fold comparing mutant to wild-type receptor. That activation ofNF-κB in transfected CHO cells by EN-RAGE resulted from ligation ofwild-type or mutant RAGE was confirmed by its inhibition in the presenceof sRAGE (FIG. 11E, lanes 8&13, respectively), or anti-RAGE IgG (FIG.11E, lanes 9&14, respectively). Incubation with nonimmune IgG had noeffect (FIG. 11E, lanes 10&15, respectively).

A critical test of these concepts was whether mononuclear phagocytes(MPs) retrieved from human subjects bearing a mutant RAGE alleledisplayed enhanced activation and generation of proinflammatorymediators in the presence of EN-RAGE. Immunoblotting revealed that basallevels of RAGE did not differ between MPs bearing wild-type (G82G),(G82S) or (S82S) alleles. Signaling was compared in MPs from patientswith RAGE (G82G) and RAGE (G82S)/(S82S) by assessing activation ofp44/p42 MAP kinases. In the presence of EN-RAGE, MPs isolated fromindividuals bearing mutant RAGE displayed an ≈4.8-fold increase inphosphorylated p44 and p42 MAP kinases compared with unstimulated cells(FIGS. 12A&B). However, MPs bearing wild-type RAGE exposed to EN-RAGErevealed a significant, although smaller (≈2.2-fold) increase inactivation of p44/p42 MAP kinases (FIGS. 12A&B). The differences betweenEN-RAGE-mediated activation of p44/p42 MAP kinases in mutant vs.wild-type RAGE-expressing MPs were significant, p<0.05 (FIG. 12B).

In order to assess the functional consequences of enhanced activation ofsignal transduction molecules stimulated upon ligation of RAGE, weexamined production of key inflammatory and tissue-degradative mediatorslinked to RA (16-19) by MPs bearing wild-type RAGE or the mutant allele.Exposure of wild-type RAGE-bearing MPs to EN-RAGE caused an ≈4-foldincrease in generation of TNF-alpha detected in culture supernatantcompared with quiescent cultures (322±51 vs 81±8.3 ng/ml; p<0.001) (FIG.12C). However, upon incubation of human MPs bearing GS or SS RAGE, an≈17.8-fold increase in elaborated TNF-alpha was observed in culturesupernatants compared to basal levels (1,623±98 vs 91±11.1 ng/ml;p<0.001) (FIG. 12C). Importantly, although basal levels of TNF-alpha didnot differ between wild-type RAGE- and mutant RAGE-bearing MPs, levelsof TNF-alpha were ≈4.5-fold more in the presence of RAGE (G82S)/(S82S)compared with wild-type RAGE, p<0.01 (FIG. 12C). Similarly, wild-typeRAGE-bearing MPs exposed to EN-RAGE displayed a small, but significant≈1.5-fold increase in generation of IL-6 compared with basal expression(29.2±4.1 vs 20.2±3.2 ng/ml; p<0.05) (FIG. 12D). However, MPs bearing GSor SS revealed an A10.9-fold augmented generation of IL-6 uponincubation with EN-RAGE compared with unstimulated controls (229.3±26.8vs 21±1.8 ng/ml; p<0.001) (FIG. 12D). Again, MPs bearing mutant RAGEgenerated increased amounts of IL-6 compared with cells from wild-typeindividuals (=7.3-fold; p<0.01) (FIG. 12D). In these studies, nosignificant differences between cellular activation induced by EN-RAGEin MPs bearing (G82S) or (S82S) RAGE were observed.

A central means by which structural elements of joints, such ascartilage and bone, are degraded in unchecked RA is by generation ofmatrix metalloproteinases (MMP), such as MMP-9. We hypothesized thatRAGE-mediated MP activation would augment generation of MMP-9 activityon cells bearing mutant receptor (G82S, S82S) versus those expressingwild-type RAGE. MPs retrieved from subjects bearing wild-type RAGEdisplayed an ≈2.3-fold increase in MMP-9 activity in the presence ofEN-RAGE compared with basal expression (p<0.01; FIGS. 12E&F). However,MPs isolated from individuals bearing RAGE (G82S)/(S82S) demonstrated an≈4.7-fold increase in EN-RAGE-mediated MMP-9 activity compared withbasal levels of expression, p<0.01. Although basal levels of MMP-9activity did not differ among GG- or GS/SS-bearing MPs, the extent ofEN-RAGE-mediated enhanced MMP-9 activity was significantly enhanced,≈2-fold, in MPs bearing mutant RAGE allele vs. wild-type receptor(p<0.01; FIGS. 12E&F).

These findings strongly suggest that in human subjects, the presence ofthe 82S allele contributes, at least in part, to enhanced susceptibilityto RA, as well as to EN-RAGE-mediated increased expression ofproinflammatory and tissue-destructive mediators highly-prevalent inrheumatoid synovium. To determine if the interaction of EN-RAGE withRAGE modulated joint inflammation and destruction in vivo, we employed amurine model of polyarticular inflammatory arthritis induced bysensitization and challenge with bovine type II collagen (10-12), thepredominant protein of articular cartilage, in dba/1 mice. Bovine typeII collagen was emulsified in incomplete Freund's adjuvant and injectedintradermally at the base of the tail (time 0; immunization). Threeweeks later, mice were challenged with a second intradermal injection ofbovine collagen type II/incomplete Freund's adjuvant (time 3 weeks;challenge). The contribution of RAGE to the pathogenesis of arthritiswas studied by treating animals with sRAGE (20). The administeredexogenous sRAGE functions as a decoy by engaging RAGE ligands andpreventing their access to cell surface receptor. Treatment with sRAGE,100 μg/day, was started at three weeks (time of challenge with bovinetype II collagen). In previous studies, blockade of RAGE at this doseaffected the greatest decrease in the proinflammatory phenotype in amurine model of delayed-type hypersensitivity (1).

The relevance of RAGE-ligand interaction in collagen-induced arthritiswas underscored by the increased expression of RAGE and EN-RAGE in jointtissues. At six weeks, compared with control mice, joint tissue from thehindpaw of mice immunized/challenged with type II collagen demonstratedhypertrophy and hyperplasia of synovial cells (FIGS. 13A&B,respectively). RAGE and EN-RAGE expression was increased in joint tissuefrom mice with arthritis compared with controls (RAGE, FIGS. 13C&D;EN-RAGE, FIGS. 13E&F, respectively). Immunoblots of joint tissue fromcontrol animals and those with arthritis to detect RAGE and EN-RAGEsshowed increased expression in each case. RAGE levels were enhanced≈2.2-fold (p<0.001) in arthritis versus control joints (FIG. 13I).Although EN-RAGEs were not detectable in joint tissue of control mice,expression of these proinflammatory mediators was induced in vehicle(murine serum albumin [MSA])-treated mice immunized/challenged withbovine type II collagen (FIG. 13J). In mice treated with sRAGE, levelsof RAGE and EN-RAGE antigen by immunoblotting were significantly reducedcompared to mice treated with MSA (FIG. 13I&J, respectively).

Consistent with the observation that blockade of RAGE in this model ofinflammatory arthritis reduced expression of RAGE andaccumulation/expression of proinflammatory EN-RAGEs, mice treated withsRAGE displayed little evidence of foot pad swelling/thickening incontrast to prominent swelling observed in MSA-treated mice evaluated atmultiple time points between 3.5-8 weeks after immunization; p<0.001(FIG. 14A). Similarly, clinical scoring of inflammatory arthritis at thewrist joint of immunized/challenged mice revealed a significantreduction in mice treated with sRAGE versus MSA (p=0.0001; FIG. 14B).

In order to dissect the molecular mechanisms underlying the apparentprotection afforded by preventing ligands from engaging cell surfaceRAGE by administration of sRAGE, we assessed plasma and joint tissuemarkers of inflammation. Immunoblots revealed undetectable TNF-alpha injoint tissue of control mice, whereas a striking induction was observedin MSA-treated mice immunized/challenged with bovine type II collagen(FIG. 14C). Animals subjected to the arthritis protocol and treated withsRAGE showed striking reduction in TNF-alpha (≈25.7-fold; p=0.001).Similarly, plasma TNF-alpha antigen, although undetectable in controlmice, was markedly induced in plasma retrieved from miceimmunized/challenged mice with type II collagen and treated with MSA(FIG. 14D). Plasma TNF-alpha was suppressed ≈2.4-fold in samples frommice treated with sRAGE (46±6.2 vs 19±1.2 ng/ml, respectively; p=0.03).IL-6 antigen in joint tissue increased in MSA-treated miceimmunized/challenged with type II collagen compared with those animalsreceiving sRAGE (1,260±465 vs 478±153 ng/μg tissue; p=0.04) (FIG. 14E).No measurable levels of IL-6 were detected in tissue retrieved fromcontrol mice. Similarly, levels of IL-2 were reduced in joint tissue ofmice treated with sRAGE (FIG. 14F).

As induction of TNF-alpha and other inflammatory cytokines sets inmotion events leading to activation of latent/proenzyme MMPs (21), weassessed MMP antigen and activity in stifle joint tissue retrieved fromthe mice employed in this model. Compared with control joint tissue,that retrieved from MSA-treated mice undergoing the collagen-inducedarthritis protocol revealed ≈2.4-fold increase in MMP-2 antigen byimmunoblotting (p=0.01; FIG. 15A). That activation of RAGE was criticalin this process was demonstrated by the significant reduction in MMP-2expression in joint tissue of sRAGE-treated mice, to levels observed inunaffected mice (p=0.02; FIG. 15A). In addition, expression of MMP-9antigen was increased ≈4.6-fold in joint tissue retrieved fromMSA-treated mice compared with animals without arthritis (p=0.01; FIG.15B). Levels of MMP-9 antigen were significantly reduced in micereceiving sRAGE compared with those mice receiving MSA; p=0.02 (FIG.15B). In order to determine the extent of activity of MMPs 2 and 9 inthe joint tissue, we performed zymography. Consistent with increasedlevels of MMP-2 and MMP-9 antigen in mice with arthritis, an ≈11.6- and≈5.5-fold increase in activity of MMP-2 and MMP-9, respectively, wasobserved in joint tissue from vehicle, MSA-treated mice compared withunafffected mice; p=0.001 and p=0.02, respectively (FIG. 15C-D). In thepresence of sRAGE, levels of MMP-2 and MMP-9 activity were reduced by≈12.2- and 4.2-fold, respectively, compared with mice receiving MSA;p=0.004 and p=0.005, respectively (FIG. 15C-D).

Lastly, in order to determine if blockade of RAGE suppressedimmune/inflammatory responses to bovine type II collagen atextra-articular sites, at 6 weeks after immunization, immediately priorto sacrifice, mice receiving either MSA or sRAGE were injected withbovine type II collagen (10 μg) into ear tissue. Although baseline earthickness was essentially identical in both groups of mice, 18 hrs afterinjection, mice receiving MSA revealed an ≈2.1-fold increase in earthickness compared with those mice injected with sRAGE (p=0.03; FIG.16A). Consistent with these observations, splenocytes retrieved fromMSA-treated mice at six weeks revealed significantly increasedproliferation, as measured by incorporation of tritiated thymidine, uponstimulation with bovine type II collagen compared with mice treated withsRAGE; p=0.003 (FIG. 16B). However, no significant differences in basallevels of proliferation, or proliferation in the presence of PMA, wereobserved between mice treated MSA vs sRAGE (FIG. 16B).

The S1000/calgranulin family of proinflammatory molecules,long-associated with classic immune/inflammatory disorders (22-23), hasbeen mechanistically linked to cellular activation resulting in aninflammatory phenotype by the observation that these molecules aresignal-transducing ligands of RAGE. Their release by activatedinflammatory effector cells, and accumulation in synovial fluid andplasma of patients with RA has been linked to indices of diseaseseverity (4), such as bony erosions. In this context, Czech subjectswith psoriasis vulgaris, an immune/inflammatory disease of the skin,displayed enrichment for the RAGE (G82S) allele compared withage-matched subjects without this skin disorder (24). Furthermore,strongly increased expression of a member of the S100 family ofproinflammatory molecules, “psoriasin”, has been demonstrated inpsoriatic lesions compared with adjacent unaffected skin (25). Theseobservations further support the premise that S100/calgranulin-RAGEinteraction may provide a mechanism contributing to immune/inflammatorydisorders. Finally, the data presented herein suggests the possibilitythat the RAGE (G82S) allele might prime affected tissues for exaggeratedinflammatory processes.

Previous studies demonstrated that the ligands of RAGE identified thusfar, each effectively cross-compete in radioligand binding assays (1).These ligands include EN-RAGE (S100A12) and related members of theS100/calgranulin family of proinflammatory cytokines (1); AdvancedGlycation Endproducts (AGEs) and, particularly, carboxy(methyl lysine)(CML) adducts of proteins and lipids (26-27); amyloid-β peptide (28);and amphoterin (15,29). Our studies support the contention that theprimary binding site for each of these ligands is within the V-domain(27), the same region in which the (G82S) substitution occurs. Indeed,substitution of glycine with serine at this site is likely to alterpolarity within that region. Consistent with this concept, wedemonstrated enhanced affinity and cellular activation mediated by oneof the receptor's ligands, EN-RAGE, on interaction with mutant RAGE(82S) compared with wild-type RAGE. Although studies to identify andcharacterize the tertiary structure of RAGE are underway, it isnevertheless certain that altered properties of ligand engagement ensuein the face of this polymorphism.

The present studies have demonstrated an association between the (G82S)RAGE polymorphism and susceptibility to RA, including those without anHLA-DR4 allele, thus providing critical evidence that our findings donot solely reflect the known linkage disequilibrium between thispolymorphism and HLA-DRB1*04 haplotypes. In the future, a detailedhaplotypic analysis of the MHC (30) may provide further evidence ofgenetic heterogeneity underlying MHC-linked susceptibility to RA.Studies are ongoing to define the precise molecular cues triggered bythis polymorphism that appear to augur enhanced susceptibility to, and,possibly, accelerated evolution of proinflammatory andtissue-degradative properties in rheumatoid synovium. Certainly,improved understanding of these complex relationships may refine notonly diagnostic criteria, but, ultimately, optimal means of therapeuticintervention in this perplexing class of human disorders.

Experimental Methods

Patient population. The rheumatoid arthritis patients used forassociation studies meet the criteria of the American College ofRheumatology (31) and were taken from patient populations (32) collectedby the North America Rheumatoid Arthritis Consortium and the ArthritisResearch Center in Wichita, Kans.

Detection of Gly82Ser polymorphism. The following primers weresynthesized for detection of the glycine82serine (G82S) polymorphism ofthe RAGE gene (8): sense primer: 5′ GTAAGCGGGGCTCCTGTTGCA-3′ (SEQ IDNO:13) and the antisense primer: 5′ GGCCAAGGCTGGGGTTGAAGG-3′ (SEQ IDNO:14). Whole blood (20 μl) was obtained from human volunteers inaccordance with the standards and policies of the Institutional ReviewBoards of the participating institutions. Genomic DNA was preparedaccording to the manufacturer's instructions using a kit from QIAGEN™(Valencia, Calif.); 10 ng was amplified using Taq I polymerase (LifeTechnologies, Grand Island, N.Y.) in a final volume of 25 μl. PCRconditions were as follows: 94° C. for 30 secs, 62° C. for 45 secs, and72° C. for 60 secs for a total of 35 cycles. PCR product (25 μl) wasthen digested with Alu 1 (Life Technologies), 3U for 16 hrs at 37° C.,followed by gel electrophoresis on agarose gels (2%).

Chinese Hamster Ovary (CHO) cell studies. Chinese hamster ovary (CHO)cells were obtained from the American Type Culture Collection (ATCC)(Manassas, Va.) and cultured in F12 medium containing fetal bovine serum(10%) (Life Technologies). In order to generate the mutant 82S allele,the cDNA encoding human RAGE33 was cloned using the TOPO TA™ cloningsystem into pCR2.1TOPO vector for mutagenesis (Invitrogen, Carlsbad,Calif.). Site-directed mutagenesis to insert the (Gly82) (wild-type) to(Ser82) (mutant) change was performed using the GENEEDITOR™ In Vitro SDMSystem (Promega, Madison, Wis.) according to the manufacturer'sinstructions. Sequencing was performed using an ABI310 automated DNAsequencer (Perkin Elmer Biosystems, Foster City, Calif.) to confirm theinserted sequence changes and to ensure that no other mutations werecreated during any of the mutagenesis reactions. Both wild-type andmutant RAGE cDNA were excised from pCR2.1TOPO using EcoR I and subclonedinto the pcDNA3.1 expression vector (Invitrogen). Cells were transfectedwith plasmid DNA using lipofectamine (Life Technologies) encoding thefollowing: pcDNA3.1 containing full-length wild-type RAGE cDNA (Gly82),pcDNA3.1 containing mutant RAGE cDNA (Ser82) or pcDNA3.1 containing noinsert (mock-transfectant). 24 hrs after transfection, selection wasbegun using G418 (1 mg/ml) (Life Technologies). RAGE expression wasassessed by immonoblotting in stably-transfected cells after 6 weeks.Cells were incubated with the indicated mediators (BSA or EN-RAGEL) andassessed for activation of phosphorylated p44/p42 MAP kinase, or fornuclear translocation of NF-kB.

Radioligand binding assays. Purified EN-RAGE was radiolabelled using¹²⁵-I and Iodobeads (Pierce, Arlington Heights, Ill.) to a specificactivity of approximately 5,000 cpm/ng. Radioligand binding assays wereperformed in 96-well tissue culture dishes containing the indicatedtransfected CHO cells. A radioligand binding assay was performed in thepresence of the indicated concentration of radiolabelled EN-RAGE±an50-fold molar excess of unlabelled EN-RAGE in PBS containingcalcium/magnesium and BSA, 0.2%, for 3 hrs at 37° C. Wells were washedrapidly with washing buffer (PBS containing Tween 20 (0.05%)). Elutionof bound material was performed in a solution containing heparin, 1mg/ml. Solution was aspirated from the wells and counted in a gammacounter (LKB, Gaithersburg, Md.). Equilibrium binding data were analyzedaccording to the equation of Klotz and Hunston (34): B=nKA/1+KA, whereB=specifically bound ligand (total binding, wells incubated with traceralone, minus nonspecific binding, wells incubated with tracer in thepresence of excess unlabeled material), n=sites/cell, K=the dissociationconstant, and A=free ligand concentration) using nonlinear least-squaresanalysis (Prism; San Diego, Calif.). Where indicated, pretreatment witheither antibodies, or human soluble RAGE, was performed.

Activation of p44/p42 MAP kinases. CHO cells were incubated withEN-RAGE, 10 μg/ml, for one hr. Cells were lysed in lysis buffer (NewEngland Biolabs, Beverly, Mass.). Cell lysate was subjected tocentrifugation and protein concentration of the supernatant determinedusing the Bio-Rad assay (Bio-Rad, Hercules, Calif.). Equal amounts ofprotein were subjected to SDS-PAGE (Novex/Invitrogen, Carlsbad, Calif.).Contents of the gels were transferred to nitrocellulose andimmunoblotting performed using anti-phosphorylated p44/p42 MAP kinase(New England Biolabs). Bands were scanned into a densitometer, and banddensity was quantified using IMAGEQUANT™ (Molecular Dynamics, FosterCity, Calif.).

Electrophoretic mobility shift assay. Nuclear extracts were prepared andEMSA performed employing consensus ³²P-labeled probe for NF-kB asdescribed (1). Where indicated, cells were treated with eithernonimmune/anti-RAGE F(ab′)2, or soluble RAGE, as described (1,20).

Peripheral Blood-Derived Mononuclear Phagocyte (MPs) Studies.

Cellular isolation. Whole venous blood was obtained from healthyvolunteers (30 ml) bearing G82G, G82S, and S82S RAGE. Mononuclear cellswere isolated using Histopaque 1077 (Sigma, St. Louis, Mo.) and culturedon plastic dishes for 3 hrs at 37° C. Nonadherent cells were removed bywashing in phosphate buffered saline (PBS). Adherent cells (MPs) wereremoved by incubation with EDTA (2 mM) for 15 mins at 37° C. Cells wereseeded in tissue-cultured coated wells for study.

Activation of p44/p42 MAP kinases. MPs were seeded into the wells of24-well tissue culture plates at a density of 5×10⁵ cells per well.Cells were stimulated with either BSA or EN-RAGE, and immunoblotting fordetection of phosphorylated p44/p42 MAP kinases performed as above.

Detection of IL-6 and TNF-alpha. MPs were seeded into the wells of24-well tissue culture plates at a density of 5×10⁵ cells per well.Cells were stimulated with either BSA or EN-RAGE (10 μg/ml). Supernatantwas assayed for IL-6 and TNF-alpha using ELISA kits from R&D systems(Minneapolis, Minn.) according to the manufacturer's instructions.

Murine studies: induction of bovine collagen type II-induced arthritis.Male dba/1 mice were purchased from the Jackson Laboratories (BarHarbor, Me.). Mice weighing 20-30 gms were injected intradermally at thebase of the tail with bovine type II collagen, 200 μg (Sigma) dissolvedin acetic acid (0.01M) and emulsified in incomplete Freund's adjuvant(Sigma). Three weeks after sensitization, mice were challenged byinjection of bovine collagen type II (200 μg) as above in incompleteFreund's adjuvant at the base of the tail. Beginning at three weeksafter immunization (at the time of challenge), mice were treated witheither murine soluble RAGE (20), or vehicle, murine serum albumin(Sigma), both at 100 μg per day by intraperitoneal injection. Treatmentwas continued daily until sacrifice.

Assessment of arthritis. Evidence of arthritis was evaluated at theindicated time points after initial immunization by an observer blindedto the experimental conditions. Severity of arthritis in the wristjoints was assessed according to the following scale: 0=no redness orswelling; 1=slight/moderate redness and swelling; and 2=severe rednessand swelling. At the same time points, extent of swelling in the distalfootpads was assessed by measurement of foot pad diameter usingcalipers. In each case, the mean of score/footpad diameter was obtainedand reported.

Injection of bovine collagen II into the ear. Six weeks after initialsensitization, bovine type II collagen (10 μg) was injected into the earof each mouse. 18 hrs later, thickness of the ear was assessed usingcalipers by an observer blinded to the experimental conditions.

Retrieval of tissues at sacrifice. Mice were sacrificed 3 or 6 weeksafter challenge. The stifle joint was removed and homogenized inTris-buffered saline containing protease inhibitors (Complete ProteaseInhibitor, Boehringer-Mannheim, Indianapolis, Ind.). From each animal,joints from the wrist and foot paw were fixed in formalin (10%) for 16hrs followed by storage in PBS for studies using hematoxylin and eosin(H&E) or the indicated antibodies.

Assessment of splenocyte proliferation. Spleens were removed atsacrifice and meshed in RPMI medium (Life Technologies) and diluted in10 ml of the same medium. The solution was subjected to centrifugationat 1,200 rpm at 4° C. for 10 mins. The pellet was dissolved in RPMImedium (15 ml) and aliquoted. Bovine type II collagen, or PMA, (0.1μg/ml in each case) was added for 24 hrs. Tritiated thymidine (0.02 ml)was then added for an additional 18 hrs. Cells were retrieved andcounted in a beta counter (LKB).

Assessment of plasma TNF-alpha. Upon sacrifice, plasma was obtained andassessed by ELISA for levels of murine TNF-alpha using a kit from R&DSystems according to the manufacturer's instructions.

Immunoblotting and ELISA. SDS-PAGE and immunoblotting were performed onextracts of stifle joint tissue using the following antibodies:anti-RAGE IgG and anti-EN-RAGE IgG as previously describedl (4.7 and 2.0μg/ml, respectively); anti-MMP 2 and anti-MMP 9 IgG (1 μg/ml; Chemicon(Temecula, Calif.); and anti-TNF-alpha IgG (1 μg/ml; R&D Systems). Bandswere scanned into a densitometer, and band density was quantified usingIMAGEQUANT™. In other experiments, assessment of joint tissue levels ofIL-6 and IL-2 was performed by subjecting joint tissue lysates to ELISAusing kits from R&D systems.

Zymography. Zymography for detection of MMP-2 and MMP-9 activity weredetermined using gelatin-laden gels from Novex/Invitrogen according tothe manufacturer instructions.

Bands were scanned into a densitometer, and band density was quantifiedas above.

Statistical analysis. Statistical comparisons among groups weredetermined using one-way analysis of variance (ANOVA); where indicated,individual comparisons were performed using students' t-test.

REFERENCES FOR EXAMPLE 4

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The shared    epitope hypothesis. An approach to understanding the molecular    genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum.    30, 1205-1213 (1987).-   6. Stastny, P. Association of the B-cell autoantigen DRW4 with    rheumatoid arthritis. N. Engl. J. Med. 298, 869-871 (1978).-   7. Sugaya, K., et al. Three genes in the MHC Class III region near    the junction with the class II: gene for Receptor of Advanced    Glycosylation End Products, PBX2 homeobox gene and a notch homolog,    human counterpart of mouse mammary tumor gene int-3. Genomics 23,    408-419 (1994).-   8. Hudson, B. I., Strickland, M. H., and Grant, P. J. Identification    of polymorphisms in the Receptor for Advanced Glycation End Products    (RAGE) gene. Diabetes 47, 1155-1157.-   9. Prevost, G., Fajardy, I., Fontaine, P., Danze, P. M., and    Besmond, C. Human RAGE Gly82Ser dimorphism and HLA class II    DRB1-DqA1-DQB1 haplotypes in type 1 diabetes. European J.    Immunogenetics 28, 343-348 (1999).-   10. Courtenay, J. S, Dallman, M. J., Dayan, A. D., Martin, A., and    Mosedale, B. Immunization against heterologous type II collagen    induces arthritis in mice. Nature 283, 666-668 (1980).-   11. Trentham, D. E., Townes, A. S., and Kang, A. H. Autoimmunity to    type II collagen an experimental model of arthritis. J. Exp. Med.    146, 857-868 (1977).-   12. Cathcart, E. S., Hayes, K. C., Gonnerman, W. A., Lazzari, A. A.,    and Franzblau, C. Experimental arthritis in a nonhuman primate. I.    Induction by bovine type II collagen. Lab. Invest. 54, 26-31 (1986).-   13. Schiff, B., Mizrachi, Y., Orgad, S., Yaron, M., and Gazit, I.    Association of HLA-Aw31 and HLA-DR1 with adult rheumatoid arthritis.    Ann. Rheum. Dis. 41, 403-406 (1991).-   14. Lander, H. L., Tauras, J. M., Ogiste, J. S., Moss, R. A.,    and A. M. Schmidt. Activation of the Receptor for Advanced Glycation    Endproducts triggers a MAP Kinase pathway regulated by oxidant    stress. J. Biol. Chem. 272, 17810-17814 (1997).-   15. Taguchi, A., et al. Blockade of amphoterin/RAGE signalling    suppresses tumor growth and metastases. Nature 405, 354-360 (2000).-   16. Pisetsky, D. S. Tumor necrosis factor blockers in rheumatoid    arthritis. N. Engl. J. Med. 342, 810-811 (2000).-   17. Boe, A., Baiocchi, M., Carbonatto, M., Papoian, R., and    Serlupi-Crescenzi, O. Interleukin-6 knock-out mice are resistant to    antigen-induced experimental arthritis. Cytokine 11, 1057-1064    (1999).-   18. Robak, T., Gladalska, A., Stepien, H., and Robak, E. Serum    levels of interleukin-6 type cytokines and soluble interleukin-6    receptor in patients with rheumatoid arthritis. Mediators Inflamm.    7, 347-353 (1998).-   19. Keyszer, G., et al. Circulating levels of matrix    metalloproteinases MMP-3 and MMP-1, tissue inhibitor of    metalloproteinases 1 (TIMP-1), and MMP-1/TIMP-1 complex in rheumatic    disease. Correlation with clinical activity of rheumatoid arthritis    versus other surrogate markers. J. Rheumatol. 26, 251-258 (1999).-   20. Park, L., et al. Suppression of accelerated diabetic    atherosclerosis by soluble Receptor for AGE (sRAGE). Nature Medicine    4, 1025-1031 (1998).-   21. Pugin, J., et al. Human neutrophils secrete gelatinase B in    vitro and in vivo in response to endotoxin and proinflammatory    mediators. Am. J. Respir. Cell. Mol. Biol. 20, 458-464 (1999).-   22. Zimmer, D. B., Cornwall, E. H., Landar, A., and Song, W. The    S100 protein family: history, function, and expression. Brain    Research Bulletin 37, 417-429 (1995).-   23. Schafer, B. W., and Heinzmann, C. W. The S100 family of EF-hand    calcium-binding proteins: functions and pathology. TIBS 21, 134-140    (1996).-   24. Kankova, K., Vasku, A., Hajek, D., Zahejsky, J., and Vasku, V.    Association of G82S polymorphism in the RAGE gene with skin    complications in type 2 diabetes. Diabetes Care 22, 1745 (1999).-   25. Madsen, P. Molecular cloning, occurrence and expression of a    novel partially secreted protein ÓpsoriasinÒ that is highly    up-regulated in psoriatic skin. J. Invest. Dermatol. 97, 701-712    (1991).-   26. Schmidt, A. M., et al. Isolation and characterization of binding    proteins for advanced glycosylation endproducts from lung tissue    which are present on the endothelial cell surface. J. Biol. Chem.    267, 14987-14997 (1992).-   27. Kislinger, T., et al. Ne (carboxymethyl)lysine modifications of    proteins are ligands for RAGE that activate cell signalling pathways    and modulate gene expression. J. Biol. Chemistry 274, 31740-31749    (1999).-   28. Yan, S. D., et al. RAGE and amyloid beta peptide neurotoxicity    in AlzheimerÕs disease. Nature 382, 685-691 (1996).-   29. Hori, O., et al. The receptor for advanced glycation endproducts    (RAGE) is a cellular binding site for amphoterin: mediation of    neurite outgrowth and coexpression of RAGE and amphoterin in the    developing nervous system. J. Biol. Chem. 270, 25752-25761 (1995).-   30. Nair, R. P., et al. Localization of psoriasis-susceptibility    locus PSORS1 to a 60-kb interval telomeric to HLA-C. Am. J. Human    Genet. 66, 1833-1844 (2000).-   31. Arnett, F. C., et al. The American Rheumatism Association 1987    revised criteria for the classification of rheumatoid arthritis.    Arthritis and Rheumatism 31, 315-324 (1988).-   32. Seldin, M. F., Amos, C. I., Ward, R., and Gregersen, P. K. The    genetics revolution and the assault on rheumatoid arthritis.    Arthritis and Rheumatism 42, 1071-1079 (1999).-   33. Neeper, M., et al. Cloning and expression of RAGE: a cell    surface receptor for advanced glycosylation end products of    proteins. J. Biol. Chem. 267, 14998-15004 (1992).-   34. 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TABLE 2 Prevalence of the RAGE 82S allele in patients with rheumatoidarthritis (RA) and controls. RA patients Controls p value All subjects76/345 (22%) 10/190 (5.3%) <0.001 DR4 negative  9/114 (7.9%)  2/141(1.4%) 0.011 subjects

Example 5 Treatment of Autoimmune Diseases as an Example of TreatingInflammation, EAE Uses of Soluble RAGE Related to Models of Autoimmune:Experimental Autoimmune Encephalitis and Adoptive Transfer Diabetes

The experiments below describe the use of soluble(s) RAGE (Receptor foradvanced glycation endproducts) to inhibit the development of autoimmune(type I) diabetes in an adoptive transfer model and the occurrence ofexperimental autoimmune encephalitis (EAE) both in murine systems.

The adoptive transfer model of diabetes involves transfer of splenocytesfrom diabetic NOD (non-obese diabetic) mice to NOD mice with severecombined immunodeficiency (scid) (1-2). The latter mice are termedNOD/scid animals, and they do not develop diabetes spontaneously.Rather, they require the presence of immunocytes capable of destroyingislet cells for induction of diabetes. This model was selected for studybecause: 1) the kinetics of disease in this model allows for a rapiddetermination of efficacy; and, 2) the model is relevant to humandisease, especially in the clinical settings in which future immunetherapies and islet transplantation is likely to occur (1-3). Thesesettings include: arresting the loss of β-cell function in individualswith new onset of Type 1 diabetes, prevention of diabetes in individualsat high risk for development of disease, and blockade of diseaserecurrence in patients with Type 1 diabetes who received islettransplants. Many immune and non-specific treatments have been found toprevent the spontaneous development of diabetes in the NOD mouse, butwith very few exceptions, these approaches have not prevented disease atits late stages or recurrent disease (3). A notable exception to thisgeneral statement includes treatment with anti-CD3 monoclonal antibodythat can prevent recurrent autoimmune diabetes in recipients of islettransplants. This drug is now in clinical trials.

The myelin basic protein (MBP) model of EAE is a widely accepted systemfor studying the pathogenesis of multiple sclerosis (4-6). Our studieshave employed the B10.PL mouse strain and two means of inducing EAE:immunization with a peptide derived from MBP and transfer of anencephalitogenic T-cell clone isolated from mice previously immunizedwith MBP (1AE10 cells). Preparation of such encephalitogenic T-cellclones is standard in the literature and has been described (7-10).Whereas the MBP immunization model provides a situation to study earlydisease, including the initial phase of sensitization to MBP, theadoptive transfer model simulates a later phase. Namely, the latter is asituation in which CD4+ lymphocytes already sensitized to MBP andactivated (i.e., fully capable of causing disease) are administered toan irradiated recipient mouse which has very limited capacity to resistthe destructive properties of the transferred immunocytes.

Methods

RAGE-blocking reagents. Two methods were used to prevent access of RAGEligands to the receptor. According to the first method, soluble RAGE(sRAGE) was prepared using recombinant DNA technology (11). The murineform of sRAGE was expressed in the baculovirus system and purified tohomogeneity based on a single band on SDS-PAGE. This material wasrequired to have an undetectable level of lipopolysaccharide using theLimulus amebocyte assay (Sigma) at an sRAGE concentration of 2 mg/ml.Polyclonal antibody to RAGE was prepared in rabbits, the IgG waspurified and characterized as described (11). Nonimmune IgG was preparedfrom rabbits not sensitized to a particular antigen. This material wassimilarly characterized for its content of lipopolysaccharide.

Adoptive transfer model of Type 1 diabetes: NOD and NOD/scid mice werepurchased from Jackson Laboratories (Bar Harbor, Me.) and housed in apathogen-free facility in the Institute for Comparative Medicine atColumbia University. Animals were monitored for development of diabetesby screening for glycosuria. Plasma glucose levels were measured in micefound to have glycosuria. Mice with diabetes (two glucose values >250mg/dl) were sacrificed by humane euthanasia and a single cell suspensionof red blood cell-depleted splenocytes was prepared. These cells weregiven intravenously (IV) to NOD/scid recipients (1.5×10⁷cells/recipient). In addition, recipients were then treated with eithersRAGE (100 μg/day, intraperitoneally [ip]) or mouse serum albumin.Plasma glucose levels were measured in capillary blood from tail veins.Mice with two values >250 mg/dl were considered to have diabetes.

Once diabetes was documented, the mice were sacrificed by humaneeuthanasia, and the pancreas was fixed in formalin and embedded inparaffin. Immunohistologic studies were then performed on histologicsections according to standard techniques (12). Antibodies employed forimmunohistologic studies were rabbit anti-tumor necrosis factor-alpha(TNF-a) IgG and rabbit anti-Interleukin (IL)-1β IgG (Santa Cruz). Theprotocol for these studies involved incubation of tissue sections withprimary antibodies (5 μg/ml) overnight at 4° C., followed by addition ofa secondary biotin-conjugated antibody (affinity purified anti-rabbitIgG; ExtrAvidin kit from Sigma). The incubation with secondary antibodywas for 30 min a 37° C., and then substrate (aminoethylcarbazole; AEC)was added (all procedures were performed according to the manufacturer'sinstructions; Sigma). Sections were counterstained with Mayer'shematoxylin. In other cases, sections were stained with hematoxylin andeosin (H&E) according to standard procedures (12).

EAE. Induction of EAE Involved Two Model Systems:

MBP immunization. A peptide comprising the N-terminal nine amino acidsof MBP (sequence: Acetylated-Ala-Ser-Gln-Arg-Lys-Pro-Ser-Gln-Arg) (SEQID NO:15) (13-14) was prepared in the Peptide Chemistry Core Laboratoryof Columbia University using standard techniques. The peptide (100μg/animal) was emulsified with complete Freund's adjuvant and injectedsubcutaneously (the total volume was 0.1 ml). Animals (B10.PL mice fromJackson Laboratory) then received two injections of pertussis toxin(total of 1 μg/mouse) intravenously (Liss Laboratories) 24 and 72 hoursafter inoculation with MBP peptide. Animals were then observed for about8 weeks for the development of symptoms of EAE. Following this period,animals were sacrificed by humane euthanasia. Where indicated, mice weretreated with sRAGE starting at the time of MBP injection. Scoring ofsymptoms was according to the following criteria (9): 0, no signs; 1,weakness of tail; 2, mild paresis of hind limbs (paraparesis); 3, severeparesis of hind limbs; 4, complete paralysis of him limbs (paraplegia)or the limbs of one side (hemiplegia); 5, death. At the time ofsymptoms, or as indicated, animals were sacrificed, and the spinal cordwas studied histologically. Spinal cord tissue was fixed in formalin,embedded in paraffin and sections were cut for H&E staining.

Transfer of an activated encephalitogenic T-cell clone. For this model,B10.PL mice were sublethally irradiated (350 R) and were then subject toadoptive transfer of an MBP-sensitized and in vitro activatedMBP-specific CD4⁺Vβ8⁺, Th1 clone termed 1AE10 (10-15×10⁶ cells/animal).The in vitro activation protocol involved culturing cells with MBPpeptide (10 μg/ml) in the presence of antigen presenting cells (theadherent population of splenocytes from B10.PL mice; 2:1 ratio ofantigen presenting cells to 1AE10 cells) for four days and addition ofIL-2 (20 U/ml; Hoffmann-LaRoche) during the last 48 hrs to increase cellnumber. Animals received intravenous pertussis toxin 24 and 72 hrs (asabove) after infusion of activated 1AE10 cells. Similar T-cell clonesand their use to induce EAE in mice have been described in theliterature (7-10). This T-cell clone has been termed 1AE10 cells. RAGEblockade was achieved using rabbit anti-RAGE IgG (50 Ag/animal/day)administered intraperitoneally) for fifteen days. Control animals weretreated identically except that nonimmune rabbit IgG was used in placeof anti-RAGE IgG. Mice were observed for 4-6 weeks for the developmentof symptoms (as above).

Results

Adoptive transfer model of diabetes. Treatment of NOD/scid recipients ofsplenocytes from diabetic NOD mice demonstrated a strong protectiveeffect of sRAGE against the development of diabetes (FIG. 17). Theislet-sparing effect of sRAGE was reversible, as discontinuance of sRAGEin 4/4 mice resulted in subsequent development of diabetes in twoseparate experiments. The latter result suggests that diabetogenicsplenocytes that had been transferred to NOD/scid recipients retainedtheir capacity to induce β-cell destruction, but, in the presence ofsRAGE, their pathogenic immune/inflammatory potential was held inabeyance.

Histology analysis of islets demonstrated a striking reduction ininflammatory infiltrates in animals treated with sRAGE compared withcontrols. Immune/inflammatory cells were consistently confined to theperiphery of islets in sRAGE-treated animals (FIGS. 18A-B).Immunohistology showed strong expression of TNF-a and IL-1β in inflamedislets from control animals after the onset of diabetes, whereassRAGE-treated animals displayed only low levels of these inflammatorymarkers constrained to the outermost periphery of islets (i.e.,peri-insulitis) (FIGS. 19A-B). Both TNF-a and IL-1β have been shown tohave direct toxic effects on β-cells (15), hence the reduced expressionof these mediators may account, at least in part, for the protectiveeffect observed in sRAGE-treated animals.

EAE models. The data shown in FIG. 20 demonstrate strongly symptomaticEAE in the vehicle-treated group, whereas sRAGE-treated mice showedsuppression of symptoms (all mice were immunized with MBP (maltosebinding protein) as described under Methods). Histologic analysis ofthese mice displayed scant infiltrates in the spinal cord ofMBP-immunized mice treated with sRAGE (FIG. 21C; this sample wasobtained on day 35 postimmunization with MBP peptide, and the mouse wasasymptomatic), compared with greater evidence of inflammatoryinfiltrates in the MBP-immunized group receiving vehicle alone (FIG. 21B[this sample was obtained 35 days after immunization with MBP and themouse had symptoms of full-blown EAE]; FIG. 21A shows a mouse notimmunized with MBP as a control). Semiquantitation of inflammatoryinfiltrates was determined by counting nuclei per high power field (10fields per slide were counted) from representative spinal cord; sectionsfrom vehicle-treated mice demonstrated a dramatic increase in nucleicoinciding with inflammatory infiltrates, whereas administration ofsRAGE caused the number of nuclei/cells per high power field to remainat the level present in normal spinal cord (FIG. 21D).

To provide a model of later-stage disease, B10.PL mice were infused with1AE10 cells. Animals developed symptoms of EAE during weeks 3-4following cell transfer whether receiving nonimmune IgG (FIG. 22) orvehicle (saline) alone (not shown). In contrast, mice treated withanti-RAGE IgG showed strong suppression of symptomatic EAE.

Discussion

The results of these studies demonstrate that blockade of RAGE, withsRAGE (which prevents access of ligands to the receptor by acting as asoluble decoy) or anti-RAGE IgG prevents the development of disease inmurine models simulating type I diabetes and multiple sclerosis (EAE).The advantage of this method is its lack of toxicity and apparenteffectiveness. An important caveat is that is difficult to be certainthat the results of our experiments can be directly extrapolated tosuccessful treatment of the human conditions. A common feature of thepathologic features of each model concerns the inability ofimmune/inflammatory cells to reach the target tissue (pancreatic isletsor spinal cord) in the presence of RAGE blockade. In the autoimmunediabetes model, this was demonstrated to be a reversible phenomenon, asstopping sRAGE resulted in the occurrence of diabetes.

REFERENCES FOR EXAMPLE 5

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1. A method for treating inflammation in a subject which comprisesadministering to the subject soluble receptor for advanced glycationendproduct (sRAGE) (SEQ ID NO:1) in an amount effective to treatinflammation in the subject.
 2. A method for treating inflammation in asubject which comprises administering to the subject a polypeptideconsisting essentially of the V-domain (SEQ ID NO:2) of receptor foradvanced glycation endproduct (RAGE) in an amount effective to treatinflammation in the subject.
 3. A method for treating inflammation in asubject which comprises administering to the subject an agent in anamount which inhibits the interaction between receptor for advancedglycation endproduct (RAGE) and its ligand thereby treating inflammationin the subject. 4-25. (canceled)